WO2021078839A1 - Animal feed composition - Google Patents

Animal feed composition Download PDF

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Publication number
WO2021078839A1
WO2021078839A1 PCT/EP2020/079715 EP2020079715W WO2021078839A1 WO 2021078839 A1 WO2021078839 A1 WO 2021078839A1 EP 2020079715 W EP2020079715 W EP 2020079715W WO 2021078839 A1 WO2021078839 A1 WO 2021078839A1
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Prior art keywords
polypeptide
seq
sequence identity
obtainable
xylanase
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PCT/EP2020/079715
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French (fr)
Inventor
Eduardo Antonio DELLA PIA
Murtala UMAR FARUK
Raffaella Aureli
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Novozymes A/S
Dsm Ip Assets B.V.
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Publication of WO2021078839A1 publication Critical patent/WO2021078839A1/en

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
    • 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
    • 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)
    • 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)
    • C12Y302/01022—Alpha-galactosidase (3.2.1.22)
    • 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)
    • C12Y302/01032—Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase
    • 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)
    • C12Y302/01136—Glucuronoarabinoxylan endo-1,4-beta-xylanase (3.2.1.136), i.e. feraxanase or feraxan-endoxylanase

Definitions

  • the present invention relates to a combination of a xylanase and an alpha-galactosidase in an animal feed for improving the nutritional value of the animal feed.
  • Xylans are hemicelluloses found in all land plants (Popper and Tuohy, 2010, Plant Physiology 153: 373-383). They are especially abundant in secondary cell walls and xylem cells. In grasses, with type II cell walls, glucurono arabinoxylans are the main hemicellulose and are present as soluble or insoluble dietary fiber in many grass based food and feed products.
  • Plant xylans have a b-1 ,4-linked xylopyranose backbone that can be substituted at the 02 or 03 position with arabinose, glucuronic acid and acetic acid in a species and tissue specific manner.
  • the starch-rich seeds of the sub-family Panicoideae with economically important species such as corn, sorghum, rice and millet have special types of highly substituted xylans in their cell walls.
  • wheat flour wherein over 60% of the xylosyl units in the arabinoxylan backbone are unsubstituted.
  • the corresponding percentage of unsubstituted backbone xylosyls is 20-30%, and in sorghum it is 35-40% (Huismann etal., 2000, Carbohydrate Polymers 42: 269-279).
  • the xylan side chains can be longer than a single arabinose or glucuronic acid substitution which is common in other xylans. This added side chain complexity is often due to L- and D-galactose and D-xylose sugars bound to the side chain arabinose or glucuronic acid.
  • the known enzymes responsible for the hydrolysis of the xylan backbone are classified into enzyme families based on sequence similarity (cazy.org).
  • the enzymes with mainly endo- xylanase activity have previously been described in Glycoside hydrolase family (GH) 5, 8, 10, 11 , 30 and 98.
  • GH Glycoside hydrolase family
  • the enzymes within a family share some characteristics such as 3D fold and they usually share the same reaction mechanism.
  • Some GH families have narrow or mono-specific substrate specificities while other families have broad substrate specificities.
  • GH10 and GH11 xylanases are often used to break down the xylose backbone of arabinoxylan. In animal feed this results in a degradation of the cereal cell wall with a subsequent improvement in nutrient release (starch and protein) encapsulated within the cells. Degradation of xylan also results in the formation of xylose oligomers that may be utilised for hind gut fermentation and therefore can help an animal to obtain more digestible energy.
  • xylanases are sensitive to side chain steric hindrance and whilst they are effective at degrading arabinoxylan from wheat, they are not very effective on the xylan found in the seeds of Poaceae species, such as corn or sorghum.
  • Soybean meal is the second biggest crop globally & the biggest protein source. Soybean meal has high protein, low fat, low starch, and high NSP content.
  • the gross energy (GE) in soybean meal (SBM) is approximately 17 MJ / kg (4000 kcal / kg).
  • poultry utilizes only approximately 55%.
  • Most of this unused energy consists of indigestible oligosaccharides and non-starch polysaccharides.
  • the indigestible soluble oligosaccharides raffinose, stachyose, verbascose
  • Oligosaccharides are also fermented in the hind-gut, causing bloating, gas production and resulting in reductions in feed intake.
  • the galactose monosaccharides are connected in a- 1.6 linkages between the galactose monomers and between galactose and sucrose.
  • Alpha-galactosidases have been selected and novel a-galactosidase have been developed to hydrolyze the complex carbohydrates of SBM and other protein meal sources, such as rapeseed, lupin, beans etc, to galactose and sucrose to increase the energy delivery from soybean meal and other feed sources.
  • Alpha-galactosidases together liberate an increased level of galactose and sucrose can be used by the animals as energy source.
  • the hydrolysis of the indigestible oligosaccharides results also in reducing their anti-nutritional effect.
  • Rovabio® Advance comprises arabinofuranosidases (ABFs), and a complex mixture of different xylanases. This large range of xylanases in combination with ABFs explains the consistency of the results regardless of the feed composition.
  • US 6,399,123 claims a method of removing oligosaccharides from feed comprising adding galactosidase, xylanase and/or cellulase to the feed in an amount of 0.1% to 1.0% by weight of the feed, hydrolyzing the oligosaccharides and then consumption by an animal.
  • US 6,399,123 further claims a method of improving BWG in chickens comprising adding a galactosidase to the feed to cause hydrolysis of the oligosaccharides and then feeding this treated feed to chickens.
  • WO 94/23022 discloses the use of a preferred alpha-galactosidase in animal feed.
  • Alpha-galactosidase is a glycoside hydrolase enzyme that hydrolyses the terminal alpha- galactosyl moieties from glycolipids and glycoproteins that is present in, e.g. legumes, vegetables, grains, cereals and the like.
  • Alpha-galactosidases are produced by various microorganisms, plants and animals, but mammals are deficient in intestinal alpha-galactosidase production and consequently are incapable of decomposing ingested alpha-galactosides by themselves. Instead, ingested alpha-galactosides are decomposed by microorganisms present in the intestine.
  • Soybean is a species of legume native to East Asia and is the second biggest feed crop globally and the biggest protein source applied in animal feed. Soybean can be manufactured (defatted) to produce soybean meal (SBM), and SBM is a significant and cheap source of high quality protein for animal feeds.
  • SBM soybean meal
  • Other common types of legume are chickpea, lupin, lentil, peanut, beans or peas which can also be processed and used as animal feed.
  • Legumes, such as soybean contain significant amounts of raffinose oligosaccharides which requires an alpha- galactosidase present to release galactose.
  • GH36 alpha-galactosidase are known in the prior art, such as disclosed in W02009/108941 (SEQ ID NO: 597, AXR38459).
  • Alpha-galactosidases and xylanases in animal feed increase the availability of the gross energy (GE) and the Average Metabolizable Energy of the animal feed by hydrolyzing the complex carbohydrates of maize, soybean meal and other protein meal sources.
  • GE gross energy
  • Metabolizable Energy of the animal feed by hydrolyzing the complex carbohydrates of maize, soybean meal and other protein meal sources.
  • the hydrolysis of the indigestible oligosaccharides by the combination results also in reducing their anti- nutritional effect.
  • One aspect of the invention is directed to a use of a combination of a polypeptide having alpha-galactosidase activity and a polypeptide having xylanase activity in an animal feed or animal feed additive, wherein the polypeptide having alpha-galactosidase activity is a GH36 alpha-galactosidase and wherein the polypeptide having xylanase activity is a GH30 xylanase.
  • a related aspect is directed to a composition comprising said combination.
  • the invention is directed to an animal feed additive comprising a polypeptide having alpha-galactosidase activity and a polypeptide having xylanase activity; wherein the polypeptide having alpha- galactosidase activity is a GH36 alpha-galactosidase and wherein the polypeptide having xylanase activity is a GH30 xylanase.
  • a further aspect of the invention is directed to a method of improving the Average Metabolizable Energy of plant-based diet in a monogastric animal comprising administering an animal feed additive, composition or the combination of the invention.
  • a further aspect is directed to an animal feed comprising the combination, the animal feed additive, or the composition of the invention, and further comprising plant-based material.
  • a further aspect is directed to a method of releasing starch from plant-based material in animal feed, comprising treating plant-based material with the combination, the animal feed additive, or the composition of the invention.
  • a further aspect is directed to a method for improving the nutritional value of an animal feed, said feed comprising plant-based material comprising adding to the feed comprising plant-based material and the combination, the animal feed additive, or the composition of the invention.
  • a further aspect is directed to a method of feeding an animal comprising adding the combination, the animal feed additive, or the composition of the invention to an animal feed.
  • SEQ ID NO: 1 is the gene sequence of GH36 alpha-galactosidase as isolated from Bacillus deramificans.
  • SEQ ID NO: 2 is the amino acid sequence as deduced from SEQ ID NO: 1.
  • SEQ ID NO: 3 is the amino acid sequence of SEQ ID NO: 2 with His-tag.
  • SEQ ID NO: 4 is the gene sequence of GH36 alpha-galactosidase as isolated from Bacillus acidopullulyticus.
  • SEQ ID NO: 5 is the amino acid sequence as deduced from SEQ ID NO: 4.
  • SEQ ID NO: 6 is the amino acid sequence of SEQ ID NO: 5 with His-tag.
  • SEQ ID NO: 7 is the gene sequence of GH36 alpha-galactosidase as isolated from Anoxybacillus bogrovensis.
  • SEQ ID NO: 8 is the amino acid sequence as deduced from SEQ ID NO: 7.
  • SEQ ID NO: 9 is the amino acid sequence of SEQ ID NO: 8 with His-tag.
  • SEQ ID NO: 10 is the gene sequence of GH36 alpha-galactosidase as isolated from Aspergillus sydowii.
  • SEQ ID NO: 11 is the amino acid sequence as deduced from SEQ ID NO: 10.
  • SEQ ID NO: 12 is the amino acid sequence of the mature GH36 alpha-galactosidase from Aspergillus sydowii.
  • SEQ ID NO: 13 is the cDNA sequence of GH36 alpha-galactosidase as isolated from Bacillus sp-19140.
  • SEQ ID NO: 14 is the amino acid sequence as deduced from SEQ ID NO: 13.
  • SEQ ID NO: 15 is the amino acid sequence of SEQ ID NO: 14 with His-tag.
  • SEQ ID NO: 16 is the corrected amino acid sequence of the alpha-galactosidase as disclosed in W01994/23022.
  • SEQ ID NO: 17 is the cDNA sequence of GH36 alpha-galactosidase as isolated from Aspergillus puniceus.
  • SEQ ID NO: 18 is the amino acid sequence as deduced from SEQ ID NO: 17.
  • SEQ ID NO: 19 is the amino acid sequence of the mature GH36 alpha-galactosidase from Aspergillus puniceus.
  • SEQ ID NO: 20 is the cDNA sequence of GH36 alpha-galactosidase as isolated from Penicillium pseudopulvillorum .
  • SEQ ID NO: 21 is the amino acid sequence as deduced from SEQ ID NO: 20.
  • SEQ ID NO: 22 is the amino acid sequence of the mature GH36 alpha-galactosidase from Penicillium pseudopulvillorum .
  • SEQ ID NO: 23 is the amino acid sequence of a mature GH30 xylanase from Bacillus subtilis.
  • SEQ ID NO: 24 is the amino acid sequence of a mature GH30 xylanase from Bacillus amyloliquefaciens.
  • SEQ ID NO: 25 is the amino acid sequence of a mature GH30 xylanase from Bacillus Hcheniformis.
  • SEQ ID NO: 26 is the amino acid sequence of a mature GH30 xylanase from Bacillus subtilis.
  • SEQ ID NO: 27 is the amino acid sequence of a mature GH30 xylanase from Paenibacillus pabuli.
  • SEQ ID NO: 28 is the amino acid sequence of a mature GH30 xylanase from Bacillus amyloliquefaciens HB-26.
  • SEQ ID NO: 29 is the amino acid sequence of a mature GH30 xylanase (as defined in WO03106654 by SEQ ID NO: 190).
  • SEQ ID NO: 30 is the amino acid sequence of the mature GH30_8 xylanase from Clostridium acetobutylicum:
  • SEQ ID NO: 31 is the amino acid sequence of the mature GH30_8 xylanase from Pseudoalteromonas tetraodonis.
  • SEQ ID NO: 32 is the amino acid sequence of the mature GH30_8 xylanase from Paenibacillus sp-19179.
  • SEQ ID NO: 33 is the amino acid sequence of the mature GH30_8 xylanase Pectobacterium carotovorum subsp. Carotovorum:
  • SEQ ID NO: 34 is the amino acid sequence of the mature GH30_8 xylanase Ruminococcus sp. CAG:330.
  • SEQ ID NO: 35 is the amino acid sequence of the mature GH30_8 xylanase Streptomyces sp-62627.
  • SEQ ID NO: 36 is the amino acid sequence of the mature GH30_8 xylanase Clostridium saccharoperb utylacetonicum .
  • SEQ ID NO: 37 is the amino acid sequence of the mature GH30_8 xylanase Paenibacillus panacisoli.
  • SEQ ID NO: 38 is the amino acid sequence of the mature GH30_8 xylanase Human Stool metagenome.
  • SEQ ID NO:39 is the amino acid sequence of the mature GH30_8 xylanase Vibrio rhizosphaerae .
  • SEQ ID NO:40 is the amino acid sequence of the mature GH30_8 xylanase from Bacillus sp-18423.
  • aspects of the invention include the use of a combination of a polypeptide having alpha- galactosidase activity and a polypeptide having xylanase activity in an animal feed or animal feed additive, wherein the polypeptide having alpha-galactosidase activity is a GH36 alpha- galactosidase and wherein the polypeptide having xylanase activity is a GH30 xylanase.
  • xylanase means a glucuronoarabinoxylan endo-1 ,4-beta-xylanase (E.C. 3.2.1.136) that catalyses the endohydrolysis of 1 ,4-beta-D-xylosyl links in some glucuronoarabinoxylans.
  • Xylanase activity can be determined with 0.2% AZCL-glucuronoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C.
  • One unit of xylanase activity is defined as 1 .0 pmole of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-glucuronoxylan as substrate in 200 mM sodium phosphate pH 6.
  • Alpha-galactosidase also called a-D-galactoside galactohydrolase (E.C. 3.2.1.22), means an enzyme that catalyses the hydrolysis of terminal, non-reducing a-D-galactose residues in a-D-galactosides, such as galactose oligosaccharides, galactomannans and galactolipids.
  • Alpha-galactosidase activity can be determined using 4- nitrophenyl a-D-galactopyranoside (available from Megazyme International, Bray, Co.
  • 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.
  • 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).
  • Arabinoxylan-containing material means any material containing arabinoxylan.
  • Arabinoxylan is a hemicellulose found in both the primary and secondary cell walls of plants, including woods and cereal grains, consisting of copolymers of two pentose sugars, arabinose and xylose.
  • the arabinoxylan chain contains a large number of 1 ,4-linked xylose units. Many xylose units are substituted with 2-, 3- or 2,3-substituted arabinose residues.
  • arabinoxylan-containing material examples include forage, roughage, seeds and grains (either whole or prepared by crushing, milling, etc from, e.g., corn, oats, rye, barley, wheat), trees or hard woods (such as poplar, willow, eucalyptus, palm, maple, birch), bamboo, herbaceous and/or woody energy crops, agricultural food and feed crops, animal feed products, cassava peels, cocoa pods, sugar cane, sugar beet, locust bean pulp, vegetable or fruit pomaces, wood waste, bark, shavings, sawdust, wood pulp, pulping liquor, waste paper, cardboard, construction and demolition wood waste, industrial or municipal waste water solids or sludge, manure, by product from brewing and/or fermentation processes, wet distillers grain, dried distillers grain, spent grain, vinasse and bagasse.
  • corn, oats, rye, barley, wheat trees or hard woods (such as poplar, willow, e
  • Forage as defined herein also includes roughage.
  • Forage is fresh plant material such as hay and silage from forage plants, grass and other forage plants, grass and other forage plants, seaweed, sprouted grains and legumes, or any combination thereof.
  • forage plants are Alfalfa (Lucerne), birdsfoot trefoil, brassica (e.g., kale, rapeseed (canola), rutabaga (swede), turnip), clover (e.g., alsike clover, red clover, subterranean clover, white clover), grass (e.g., Bermuda grass, brome, false oat grass, fescue, heath grass, meadow grasses, miscanthus, orchard grass, ryegrass, switchgrass, Timothy-grass), corn (maize), hemp, millet, barley, oats, rye, sorghum, soybeans and wheat and vegetables such as beets.
  • Crops suitable for ensilage are the ordinary grasses, clovers, alfalfa, vetches, oats, rye and maize.
  • Forage further includes crop residues from grain production (such as corn stover; straw from wheat, barley, oat, rye and other grains); residues from vegetables like beet tops; residues from oilseed production like stems and leaves form soy beans, rapeseed and other legumes; and fractions from the refining of grains for animal or human consumption or from fuel production or other industries.
  • Roughage is generally dry plant material with high levels of fiber, such as fiber, bran, husks from seeds and grains and crop residues (such as stover, copra, straw, chaff, sugar beet waste).
  • Preferred sources of arabinoxylan-containing materials are forage, roughage, seeds and grains, sugar cane, sugar beet and wood pulp.
  • 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.
  • 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 (i.e., from the same gene) or foreign (i.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.
  • 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.
  • 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 feed 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 polypeptide; wherein the fragment has xylanase activity.
  • a fragment comprises at least 330 amino acid residues, at least 350 amino acid residues, or at least 370 amino acid residues.
  • Highly branched xylan means that more than 50% of xylosyl units in the arabinoxylan backbone are substituted. This is preferably calculated from linkage analysis as performed in Huismann et al. Carbohydrate Polymers, 2000, 42:269-279.
  • Host cell means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.
  • 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.
  • Improved property means a characteristic associated with a variant that is improved compared to a parent.
  • Such improved properties include, but are not limited to, catalytic efficiency, catalytic rate, chemical stability, oxidation stability, pH activity, pH stability, specific activity, stability under storage conditions, substrate binding, substrate cleavage, substrate specificity, substrate stability, surface properties, thermal activity, and thermostability.
  • the improved property is improved thermostability.
  • 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.
  • a host cell may produce a mixture of two of more different mature polypeptides (i.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 having xylanase activity.
  • Mutant means a polynucleotide encoding a variant.
  • 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 digesta 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 digesta 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.
  • operably 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 CaCI , pH 5 and allowed to hydrate for 30 min at room temperature under gently stirring. After hydration, 200 pi substrate suspension was 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). The enzyme/substrate mixtures were left for hydrolysis in 2.5 h at 40°C under gently agitation (500 RPM) in a plate incubator.
  • the enzyme/substrate plates were centrifuged for 10 min at 3000 RPM and 50 mI supernatant was mixed with 100 mI 1.6 M HCI and transferred to 300 mI PCR tubes and left for acid hydrolysis for 40 min at 90°C in a PCR machine. Samples were neutralized with 125 mI 1.4 M NaOH after acid hydrolysis and loaded on the HPAE-PAD for mono-saccharide analysis.
  • Sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.
  • the 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, 2000, Trends Genet. 16: 276-277), 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 EBLOSUM62 (EMBOSS version of BLOSUM62) 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:
  • sequence identity between two 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:
  • Subsequence means a polynucleotide having one or more (e.g., several) nucleotides absent from the 5' and/or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having xylanase activity.
  • 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 + Ser411Phe” or “G205R + S411F”, representing substitutions at positions 205 and 411 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively.
  • + addition marks
  • 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 polypeptide having xylanase activity are xylanases of the GH30 family, preferably GH30 subfamily 8 variants.
  • the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; q) a polypeptide having at least 90% sequence identity to SEQ ID NO: 24; r) a polypeptide having at least 90% sequence identity to SEQ ID NO: 25; s) a polypeptide having at least 90% sequence identity to SEQ ID NO: 26; t) a polypeptide having at least 90% sequence identity to SEQ ID NO: 27; u) a polypeptide having at least 90% sequence identity to SEQ ID NO: 28; v) a polypeptide having at least 90% sequence identity to SEQ ID NO: 29; w) a polypeptide having at least 90% sequence identity to SEQ ID NO: 30; x
  • the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide obtained or obtainable from Bacillus subtilis having at least 90% sequence identity to SEQ ID NO: 23; q) a polypeptide obtained or obtainable from Bacillus amyloliquefaciens having at least 90% sequence identity to SEQ ID NO: 24; r) a polypeptide obtained or obtainable from Bacillus Hcheniformis having at least 90% sequence identity to SEQ ID NO: 25; s) a polypeptide obtained or obtainable Bacillus subtilis having at least 90% sequence identity to SEQ ID NO: 26; t) a polypeptide obtained or obtainable from Paenibacillus pabuli having at least 90% sequence identity to SEQ ID NO: 27; u) a polypeptide obtained or obtainable from Bacillus amyloliquefaciens having at least 90% sequence identity to SEQ ID NO: 28; v
  • the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; u) a polypeptide having at least 90% sequence identity to SEQ ID NO: 29; and gg) a polypeptide having at least 90% sequence identity to SEQ ID NO: 40.
  • the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; and gg) a polypeptide having at least 90% sequence identity to SEQ ID NO: 40, such as wherein polypeptide having xylanase activity is a GH30 xylanase having at least 90% sequence identity to SEQ ID NO: 23.
  • the xylanase of the combination of the invention may be, in one embodiment , selected from SEQ ID NO: 29, and comprising a substitution at one or more (e.g., several) positions corresponding to positions 24, 26, 36, 37, 60, 71 , 74, 75, 76, 124, 133, 155, 167, 208, 317, and 321 of SEQ ID NO: 29, wherein the variant has xylanase activity and wherein the variant has xylanase activity and has at least 70% identity, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, or most preferably at least 95% identity to SEQ ID NO: 29.
  • the polypeptide having xylanase activity has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 23.
  • the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 24.
  • the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 25.
  • the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 26.
  • the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 27.
  • the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 28.
  • the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 29.
  • the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO:30.
  • the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 31.
  • the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 32.
  • the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 33.
  • the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 34.
  • the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 35.
  • the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 36.
  • the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 37.
  • the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 38.
  • the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 39.
  • the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 40.
  • the xylanase comprises or consists of the substitutions H24W + A26E of the polypeptide of SEQ ID NO: 29. In another embodiment, the xylanase comprises or consists of the substitutions H24W + V74L of the polypeptide of SEQ ID NO: 29.
  • the xylanase comprises or consists of the substitutions H24W + K75L of the polypeptide of SEQ ID NO: 29.
  • the xylanase comprises or consists of the substitutions H24W + H76L of the polypeptide of SEQ ID NO: 29.
  • the xylanase comprises or consists of the substitutions H24W + I155M of the polypeptide of SEQ ID NO: 29.
  • the xylanase comprises or consists of the substitutions H24W + V208L of the polypeptide of SEQ ID NO: 29.
  • the xylanase comprises or consists of the substitutions A26E + V74L of the polypeptide of SEQ ID NO: 29.
  • the xylanase comprises or consists of the substitutions A26E + K75L of the polypeptide of SEQ ID NO: 29.
  • the xylanase comprises or consists of the substitutions A26E + H76L of the polypeptide of SEQ ID NO: 29.
  • the xylanase comprises or consists of the substitutions A26E + I155M of the polypeptide of SEQ ID NO: 29.
  • the xylanase comprises or consists of the substitutions A26E + V208L of the polypeptide of SEQ ID NO: 29.
  • the xylanase comprises or consists of the substitutions V74L + K75L of the polypeptide of SEQ ID NO: 29.
  • the xylanase comprises or consists of the substitutions V74L + H76L of the polypeptide of SEQ ID NO: 29.
  • the xylanase comprises or consists of the substitutions V74L + I155M of the polypeptide of SEQ ID NO: 29.
  • the xylanase comprises or consists of the substitutions V74L + V208L of the polypeptide of SEQ ID NO: 29.
  • the xylanase comprises or consists of the substitutions K75L + H76L of the polypeptide of SEQ ID NO: 29.
  • the xylanase comprises or consists of the substitutions K75L + I155M of the polypeptide of SEQ ID NO: 29.
  • the xylanase comprises or consists of the substitutions K75L + V208L of the polypeptide of SEQ ID NO: 29.
  • the xylanase comprises or consists of the substitutions H76L + I155M of the polypeptide of SEQ ID NO: 29. In another embodiment, the xylanase comprises or consists of the substitutions H76L + V208L of the polypeptide of SEQ ID NO: 29.
  • the xylanase comprises or consists of the substitutions I155M + V208L of the polypeptide of SEQ ID NO: 29.
  • the xylanase may further comprise one or more additional alterations at one or more ( e.g ., several) other positions.
  • the xylanase may further comprise one or more amino acid substitutions.
  • the 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 1-50 amino acids such as 1-40 amino acids or 1-30 amino acides; 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.
  • G to A A to G, S; V to I, L, A, T, S; I to V, L, M; L to I, M, V; M to L, I, V; P to A, S, N; F to Y, W, H; Y to F, W, H; W to Y, F, H; R to K, E, D; K to R, E, D; H to Q, N, S; D to N, E, K, R, Q; E to Q, D, K, R, N; S to T, A; T to S, V, A; C to S, T, A; N to D, Q, H, S; Q to E, N, H, K, R.
  • amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered.
  • amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.
  • 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 mutant molecules are tested for xylanase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et ai., 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, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et at, 1992, Science 255: 306-312; Smith et a!., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et at, 1992, FEBS Lett. 309: 59-64.
  • the identity of essential amino acids can also be inferred from an alignment with a related polypeptide.
  • the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 23; q) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 24; r) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 25; s) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 26; t) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 27; u)
  • the xylanase is obtained or obtainable from the taxonomic order Bacillales, preferably the taxonimic family Bacillaceae, or more preferably from the genus Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis or Paenibacillus pabuli.
  • the xylanase is obtained or obtainable from the taxonomic order Bacillales, preferably the taxonimic family.
  • theparent xylanase is a GH30 subfamily 8 xylanase.
  • 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. Biotechnol. 76: 245-251 ; Rasmussen- Wilson et al., 1997, Appl. Environ. Microbiol.
  • 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 xylanse encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted.
  • the xylanase is secreted extracellularly.
  • the polypeptide may be a bacterial polypeptide.
  • the polypeptide may be a Gram-positive bacterial polypeptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces polypeptide having xylanase activity.
  • the polypeptide is from a bacterium of the class Bacilli, such as from the order Bacillales, or from the family Paenibacillaceae, or from the genus Paenibacillus or from the species Paenibacillus sp-19179 or Paenibacillus panacisoli.
  • the polypeptide is from a bacterium of the class Clostridia, such as from the order Clostridiales, or from the family Clostridiaceae, or from the genus Clostridium or from the species Clostridium saccharobutylicum.
  • Clostridia such as from the order Clostridiales, or from the family Clostridiaceae, or from the genus Clostridium or from the species Clostridium saccharobutylicum.
  • the polypeptide is from a bacterium of the class Clostridia, such as from the order Clostridiales, or from the family Ruminococcaceae, or from the genus Ruminococcus, or from the species Ruminococcus sp. CAG:330.
  • polypeptide is from a bacterium of the class
  • Gammaproteobacteria such as from the order Alteromonadales, or from the family Pseudoalteromonadaceae, or from the genus Pseudoalteromonas or from the species Pseudoalteromonas tetraodonis.
  • polypeptide is from a bacterium of the class
  • Gammaproteobacteria such as from the order Enterobacteriales, or from the family Enterobacteriaceae, or from the genus Pectobacterium or from the species Pectobacterium carotovorum.
  • the polypeptide is from a bacterium of the class Actinobacteria, such as from the order Streptomycetales, or from the family Streptomycetaceae, or from the genus Streptomyces or from the species Streptomyces sp-62627.
  • polypeptide is from a bacterium of the class
  • Gammaproteobacteria such as from the order Vibrionales, or from the family Vibrionaceae, or from the genus Vibrio or from the species Vibrio rhizosphaerae.
  • the xylanse is a Bacillus al alophilus, 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, or Bacillus thuringiensis xylanase.
  • the xylanase is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus xylanase.
  • the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents. Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
  • 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 polypeptide having alpha-galactosidase activity of the present invention 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 polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted.
  • the polypeptide obtained from a given source is secreted extracellularly.
  • the polypeptide is from a bacterium of the class Bacilli, such as from the order Bacillales, or from the family Bacillaceae, or from the genus Bacillus or from the species Bacillus acidopullulyticus, Bacillus sp-19140 or Bacillus deramificans.
  • the polypeptide is from a bacterium of the class Bacilli, such as from the order Bacillales, or from the family Bacillaceae, or from the genus Anoxybacillus or from the species Anoxybacillus bogrovensis.
  • the polypeptide is from a fungus of the class Eurotiomycetes, such as from the order Eurotiales, or from the family Aspergillaceae, or from the genus Aspergillus or from the species Aspergillus unguis or Aspergillus puniceus.
  • the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
  • 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. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), 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 invention relates to a use of a combination of a polypeptide having alpha- galactosidase activity and a polypeptide having xylanase activity in an animal feed or animal feed additive, wherein the polypeptide having alpha-galactosidase activity is a GH36 alpha- galactosidase and wherein the polypeptide having xylanase activity is a GH30 xylanase.
  • the polypeptide having alpha-galactosidase activity is a GH36 alpha-galactosidase typically selected from the group consisting of a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide having at least 90% sequence identity to SEQ ID NO: 9; g) a polypeptide having at least 90% sequence identity to SEQ ID NO: 11 ; h) a polypeptide having at least 90% sequence identity to SEQ ID NO: 12; i) a polypeptide having at least 90% sequence identity to SEQ ID NO: 14; j) a polypeptide having at least 90% sequence identity
  • the polypeptide having alpha-galactosidase activity is a GH36 alpha- galactosidase selected from the group consisting of a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide having at least 90% sequence identity to SEQ ID NO: 9; k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; n) a polypeptide having at least 90% sequence identity to SEQ ID NO: 21 ; and o) a polypeptide having at least 90% sequence identity
  • the polypeptide having alpha-galactosidase activity is a polypeptide selected from the group consisting of k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO: 22
  • the polypeptide having alpha-galactosidase activity is a GH36 alpha-galactosidase typically selected from the group consisting of a) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 2; b) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 3; c) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 5; d) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to S
  • composition comprises the polypeptides of the invention and one or more formulating agents, as described below.
  • compositions 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-mannosidase (
  • compositions may further comprise one or more microbes.
  • the microbe is selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium sp., Carnobacterium sp., Clostridium butyricum, Clostridium sp., Enterococcus faecium, Enterococcus sp., Lactobacillus sp., Lactobacillus acidophilus, Lactobacillus farciminus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, Lactococcus lactis, Lactococcoccus
  • the 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, dextri
  • the composition comprises one or more components selected from the list consisting of vitamins, minerals and amino acids.
  • the composition comprises plant based material from the sub-family Panicoideae as disclosed herein, preferably maize, corn, sorghum, switchgrass, millet, pearl millet, foxtail millet or in a processed form such as milled corn, milled maize, defatted maize, defatted destarched maize, milled sorghum, milled switchgrass, milled millet, milled foxtail millet, milled pearl millet, or any combination thereof.
  • the enzyme combination of the invention may be formulated as a liquid or a solid.
  • the formulating agent may comprise a polyol (such as e.g. glycerol, ethylene glycol or propylene glycol), a salt (such as e.g. sodium chloride, sodium benzoate, potassium sorbate) or a sugar or sugar derivative (such as e.g. dextrin, glucose, sucrose, and sorbitol).
  • a polyol such as e.g. glycerol, ethylene glycol or propylene glycol
  • a salt such as e.g. sodium chloride, sodium benzoate, potassium sorbate
  • a sugar or sugar derivative such as e.g. dextrin, glucose, sucrose, and sorbitol
  • the composition is a liquid composition
  • the polypeptide of the invention and one or more formulating agents selected from the list consisting of glycerol, ethylene glycol, 1 ,2-propylene glycol, 1 ,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, dextrin, glucose, sucrose, and sorbitol.
  • the liquid formulation may be sprayed onto the feed after it has been pelleted or may be added to drinking water given to the animals.
  • the formulation may be for example as a granule, spray dried powder or agglomerate (e.g. as disclosed in W02000/70034).
  • the formulating agent may comprise a salt (organic or inorganic zinc, sodium, potassium or calcium salts such as e.g.
  • a sugar or sugar derivative such as e.g. sucrose, dextrin, glucose, lactose, sorbitol
  • the composition is a solid composition, such as a spray dried composition, comprising the xylanase of the invention and one or more formulating agents selected from the list consisting of sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch and cellulose.
  • the formulating agent is selected from one or more of the following compounds: sodium sulfate, dextrin, cellulose, sodium thiosulfate, magnesium sulfate and calcium carbonate.
  • the present invention also relates to enzyme granules/particles comprising the combination of the invention optionally combined with one or more additional enzymes.
  • the granule is composed of a core, and optionally one or more coatings (outer layers) surrounding the core.
  • the granule/particle size, measured as equivalent spherical diameter (volume based average particle size), of the granule is 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.
  • the core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.
  • Preparation methods include known feed and granule formulation technologies, e.g. : a) spray dried products, wherein a liquid enzyme-containing solution is atomized in a spray drying tower to form small droplets which during their way down the drying tower dry to form an enzyme-containing particulate material; b) layered products, wherein the enzyme is coated as a layer around a pre-formed inert core particle, wherein an enzyme-containing solution is atomized, typically in a fluid bed apparatus wherein the pre-formed core particles are fluidized, and the enzyme-containing solution adheres to the core particles and dries up to leave a layer of dry enzyme on the surface of the core particle.
  • Particles of a desired size can be obtained this way if a useful core particle of the desired size can be found.
  • This type of product is described in, e.g., WO 97/23606; c) absorbed core particles, wherein rather than coating the enzyme as a layer around the core, the enzyme is absorbed onto and/or into the surface of the core.
  • a process is described in WO 97/39116; d) extrusion or pelletized products, wherein an enzyme-containing paste is pressed to pellets or under pressure is extruded through a small opening and cut into particles which are subsequently dried.
  • Such particles usually have a considerable size because of the material in which the extrusion opening is made (usually a plate with bore holes) sets a limit on the allowable pressure drop over the extrusion opening. Also, very high extrusion pressures when using a small opening increase heat generation in the enzyme paste, which is harmful to the enzyme; e) prilled products, wherein an enzyme-containing powder is suspended in molten wax and the suspension is sprayed, e.g., through a rotating disk atomiser, into a cooling chamber where the droplets quickly solidify (Michael S. Showell (editor); Powdered detergents ⁇ , Surfactant Science Series; 1998; vol. 71 ; page 140-142; Marcel Dekker).
  • the product obtained is one wherein the enzyme is uniformly distributed throughout an inert material instead of being concentrated on its surface.
  • US 4,016,040 and US 4,713,245 are documents relating to this technique; f) mixer granulation products, wherein a liquid is added to a dry powder composition of, e.g., conventional granulating components, the enzyme being introduced either via the liquid or the powder or both. The liquid and the powder are mixed and as the moisture of the liquid is absorbed in the dry powder, the components of the dry powder will start to adhere and agglomerate and particles will build up, forming granulates comprising the enzyme.
  • the cores comprising the enzyme contain a low amount of water before coating. If water sensitive enzymes are coated before excessive water is removed, it will be trapped within the core and it may affect the activity of the enzyme negatively.
  • the cores preferably contain 0.1-10 % w/w water.
  • the core may include additional materials such as fillers, fibre materials (cellulose or synthetic fibres), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.
  • additional materials such as fillers, fibre materials (cellulose or synthetic fibres), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.
  • the core may include a binder, such as synthetic polymer, wax, fat, or carbohydrate.
  • a binder such as synthetic polymer, wax, fat, or carbohydrate.
  • the core may include a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and/or an acidic buffer component, typically as a homogenous blend.
  • the core comprises a material selected from the group consisting of salts (such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (such as e.g.
  • salts such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate,
  • sucrose, dextrin, glucose, lactose, sorbitol sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol), small organic molecules, starch, flour, cellulose and minerals and clay minerals (also known as hydrous aluminium phyllosilicates).
  • the core comprises a clay mineral such as kaolinite or kaolin.
  • the core may include an inert particle with the enzyme absorbed into it, or applied onto the surface, e.g., by fluid bed coating.
  • the core may have a diameter of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.
  • the core may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule.
  • the optional coating(s) may include a salt and/or wax and/or flour coating, or other suitable coating materials.
  • the coating may be applied in an amount of at least 0.1% by weight of the core, e.g., at least 0.5%, 1% or 5%.
  • the amount may be at most 100%, 70%, 50%, 40% or 30%.
  • the coating is preferably at least 0.1 pm thick, particularly at least 0.5 pm, at least 1 pm or at least 5 pm. In some embodiments the thickness of the coating is below 100 pm, such as below 60 pm, or below 40 pm.
  • the coating should encapsulate the core unit by forming a substantially continuous layer.
  • a substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit is encapsulated or enclosed with few or no uncoated areas.
  • the layer or coating should in particular be homogeneous in thickness.
  • the coating can further contain other materials as known in the art, e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.
  • fillers e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.
  • a salt coating may comprise at least 60% by weight of a salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.
  • the salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles are less than 50 pm, such as less than 10 pm or less than 5 pm.
  • the salt coating may comprise a single salt or a mixture of two or more salts.
  • the salt may be water soluble, in particular having a solubility at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g per 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 g per 100 g water.
  • the salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate or acetate.
  • simple organic acids e.g., 6 or less carbon atoms
  • Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminium.
  • anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, sorbate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate.
  • alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used.
  • the salt in the coating may have a constant humidity at 20°C above 60%, particularly above 70%, above 80% or above 85%, or it may be another hydrate form of such a salt (e.g., anhydrate).
  • the salt coating may be as described in W01997/05245, W01998/54980, W01998/55599, W02000/70034, W02006/034710, W02008/017661 , W02008/017659,
  • NaH 2 P0 4 , (NH4)H 2 P0 4 , CuS0 4 , Mg(N0 3 ) 2 magnesium acetate, calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, sodium acetate, sodium benzoate, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate and zinc sorbate.
  • the salt may be in anhydrous form, or it may be a hydrated salt, i.e. a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99/32595.
  • Specific examples include anhydrous sodium sulfate (Na 2 S0 4 ), anhydrous magnesium sulfate (MgS0 4 ), magnesium sulfate heptahydrate (MgS0 4 .7H 2 0), zinc sulfate heptahydrate (ZnS0 4 .7H 2 0), sodium phosphate dibasic heptahydrate (Na 2 HP0 4 .7H 2 0), magnesium nitrate hexahydrate (Mg(N0 3 ) 2 (6H 2 0)), sodium citrate dihydrate and magnesium acetate tetrahydrate.
  • the salt is applied as a solution of the salt, e.g., using a fluid bed.
  • a wax coating may comprise at least 60% by weight of a wax, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.
  • waxes are polyethylene glycols; polypropylenes; Carnauba wax; Candelilla wax; bees wax; hydrogenated plant oil or animal tallow such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC), polyvinyl alcohol (PVA), hydrogenated ox tallow, hydrogenated palm oil, hydrogenated cotton seeds and/or hydrogenated soy bean oil; fatty acid alcohols; mono-glycerides and/or di-glycerides, such as glyceryl stearate, wherein stearate is a mixture of stearic and palmitic acid; micro-crystalline wax; paraffin’s; and fatty acids, such as hydrogenated linear long chained fatty acids and derivatives thereof.
  • a preferred wax is palm oil or hydrogenated palm oil.
  • the granule may comprise a core comprising the xylanase of the invention, one or more salt coatings and one or more wax coatings.
  • a core comprising the xylanase of the invention, one or more salt coatings and one or more wax coatings. Examples of enzyme granules with multiple coatings are shown in WO1993/07263, WO1997/23606 and WO2016/149636.
  • Non-dusting granulates may be produced, e.g., as disclosed in U.S. Patent Nos. 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art.
  • the coating materials can be waxy coating materials and film-forming coating materials.
  • waxy coating materials are polyethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
  • film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591.
  • the granulate may further comprise one or more additional enzymes. Each enzyme will then be present in more granules securing a more uniform distribution of the enzymes, and also reduces the physical segregation of different enzymes due to different particle sizes. Methods for producing multi-enzyme co-granulates is disclosed in the ip.com disclosure IPCOM000200739D.
  • the present invention also relates to protected enzymes prepared according to the method disclosed in EP 238,216.
  • the present invention provides a granule, which comprises:
  • the coating comprises a salt coating as described herein. In one embodiment, the coating comprises a wax coating as described herein. In one embodiment, the coating comprises a salt coating followed by a wax coating as described herein.
  • An animal feed additive comprising a polypeptide having alpha-galactosidase activity and a polypeptide having xylanase activity; wherein the polypeptide having alpha-galactosidase activity is a GH36 alpha-galactosidase and wherein the polypeptide having xylanase activity is a GH30 xylanase.
  • the animal feed additive comprises a polypeptide having alpha-galactosidase activity wherein the GH36 alpha-galactosidase selected from the group consisting of a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide having at least 90% sequence identity to SEQ ID NO: 9; g) a polypeptide having at least 90% sequence identity to SEQ ID NO: 11 ; h) a polypeptide having at least 90% sequence identity to SEQ ID NO: 12; i) a polypeptide having at least 90% sequence identity to SEQ ID NO: 14;
  • the animal feed additive comprises a polypeptide having alpha-galactosidase activity wherein the GH36 alpha-galactosidase selected from the group consisting of a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide having at least 90% sequence identity to SEQ ID NO: 9; k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; n) a polypeptide having at least 90% sequence identity to SEQ ID NO: 21 ; and o) a polypeptide
  • the animal feed additive comprises a polypeptide having alpha-galactosidase activity wherein the GH36 alpha-galactosidase selected from the group consisting of excellent) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO: 22 and wherein the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; u) a polypeptide having at least 90% sequence identity to SEQ ID NO: 29; and gg) a polypeptide having at least 90% sequence identity to SEQ ID NO: 40.
  • the animal feed additive comprises wherein a polypeptide having alpha-galactosidase activity selected from k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; and a polypeptide having xylanase activity selected from p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23.
  • the animal feed additive may further comprise one or more additional components selected from the group consisting of: one or more vitamins; one or more minerals; one or more amino acids; one or more phytogenies; one or more prebiotics; one or more organic acids; and one or more other feed ingredients.
  • the animal feed additive typically comprises an amount of alpha-galactosidase and xylanase so as to increase the Average Metabolizable Energy of plant-based diet.
  • An interesting aspect of the invention is directed to a method of improving the Average Metabolizable Energy of plant-based diet in a monogastric animal comprising administering an animal feed additive, composition, or combination as defined herein.
  • the animal feed or animal feed additive comprises a formulating agent and the enzyme combination of the invention.
  • the formulating agent comprises one or more of the following compounds: 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 and cellulose.
  • the invention further relates to an animal feed additive optionally comprising one or more vitamins and the enzyme combination of the invention.
  • the invention also relates to an animal feed additive comprising one or more minerals and the enzyme combination of the invention.
  • the invention also relates to an animal feed additive comprising one or more amino acids and a xylanase variant of the invention.
  • the amount of each of the enzymes in the animal feed additive is between 0.001% and 10% by weight of the composition.
  • the animal feed additive comprises one or more formulating agents, preferably as described herein above.
  • the animal feed additive comprises one or more additional enzymes, preferably as described herein below.
  • the animal feed additive comprises one or more probiotics, preferably as described herein below.
  • the animal feed additive comprises one or more vitamins, preferably as described herein below.
  • the animal feed additive comprises one or more minerals, preferably as described herein below.
  • the animal feed additive comprises one or more amino acids, preferably as described herein below.
  • the animal feed additive comprises one or more prebiotics, preferably as described herein below. In an embodiment, the animal feed additive comprises one or more organic acids, preferably as described herein below.
  • the animal feed additive comprises one or more phytogenies, preferably as described herein below.
  • the present invention also relates to animal feed compositions comprising the combination or animal feed of the invention.
  • the invention also relates to an animal feed comprising the granule as described herein and plant based material.
  • the invention also relates to an animal feed comprising the animal feed additive as described herein and plant based material.
  • the plant based material is from the sub-family Panicoideae.
  • Animal feed compositions or diets have a relatively high content of protein.
  • Poultry and pig diets can be characterised as indicated in Table B of WO 01/58275, columns 2-3.
  • Fish diets can be characterised as indicated in column 4 of this Table B. Furthermore such fish diets usually have a crude fat content of 200-310 g/kg.
  • An animal feed composition according to the invention has a crude protein content of 50- 800 g/kg, and furthermore comprises at least one xylanase as claimed herein.
  • the animal feed composition of the invention has a content of metabolisable energy of 10-30 MJ/kg; and/or a content of calcium of 0.1-200 g/kg; and/or a content of available phosphorus of 0.1-200 g/kg; and/ora content of methionine of 0.1-100 g/kg; and/or a content of methionine plus cysteine of 0.1-150 g/kg; and/or a content of lysine of 0.5-50 g/kg.
  • the content of metabolisable energy, crude protein, calcium, phosphorus, methionine, methionine plus cysteine, and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO 01/58275 (R. 2-5).
  • the nitrogen content is determined by the Kjeldahl method (A.O.A.C., 1984, Official Methods of Analysis 14th ed., Association of Official Analytical Chemists, Washington DC).
  • Metabolisable energy can be calculated on the basis of the NRC publication Nutrient requirements in swine, ninth revised edition 1988, subcommittee on swine nutrition, committee on animal nutrition, board of agriculture, national research council. National Academy Press, Washington, D.C., pp. 2-6, and the European Table of Energy Values for Poultry Feed-stuffs, Spelderholt centre for poultry research and extension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen & looijen bv, Wageningen. ISBN 90-71463-12-5.
  • the dietary content of calcium, available phosphorus and amino acids in complete animal diets is calculated on the basis of feed tables such as Veevoedertabel 1997, gegevens over chemische samenstelling, verteerbaarheid en voederwaarde van voedermiddelen, Central Veevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.
  • the animal feed composition of the invention contains at least one vegetable protein as defined above.
  • the animal feed composition of the invention may also contain animal protein, such as Meat and Bone Meal, Feather meal, and/or Fish Meal, typically in an amount of 0-25%.
  • animal feed composition of the invention may also comprise Dried Distillers Grains with Solubles (DDGS), typically in amounts of 0-30%.
  • DDGS Dried Distillers Grains with Solubles
  • the animal feed composition of the invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or 0-20% whey.
  • the animal feed may comprise vegetable proteins.
  • the protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (w/w).
  • Vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example, materials from plants of the families Fabaceae ( Leguminosae ), Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupin meal, rapeseed meal, and combinations thereof.
  • the vegetable protein source is material from one or more plants of the family Fabaceae, e.g., soybean, lupine, pea, or bean.
  • the vegetable protein source is material from one or more plants of the family Chenopodiaceae, e.g. beet, sugar beet, spinach or quinoa.
  • Other examples of vegetable protein sources are rapeseed, and cabbage.
  • soybean is a preferred vegetable protein source.
  • Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, and sorghum.
  • Animal diets can e.g. be manufactured as mash feed (non-pelleted) or pelleted feed.
  • the milled feed-stuffs are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species in question.
  • Enzymes can be added as solid or liquid enzyme formulations.
  • mash feed a solid or liquid enzyme formulation may be added before or during the ingredient mixing step.
  • pelleted feed the (liquid or solid) xylanase/enzyme preparation may also be added before or during the feed ingredient step.
  • a liquid xylanase/enzyme preparation comprises the xylanase of the invention optionally with a polyol, such as glycerol, ethylene glycol or propylene glycol, and is added after the pelleting step, such as by spraying the liquid formulation onto the pellets.
  • a polyol such as glycerol, ethylene glycol or propylene glycol
  • the enzyme may also be incorporated in a feed additive or premix.
  • the xylanase can be prepared by freezing a mixture of liquid enzyme solution with a bulking agent such as ground soybean meal, and then lyophilizing the mixture.
  • the final enzyme concentration in the diet is within the range of 0.01-200 mg enzyme protein per kg diet, preferably between 0.05-100 mg/kg diet, more preferably 0.1-50 mg, even more preferably 0.2-20 mg enzyme protein per kg animal diet.
  • the enzyme is administered in one or more of the following amounts (dosage ranges): 0.01-200; 0.05-100; 0.1-50; 0.2-20; 0.1-1 ; 0.2-2; 0.5-5; or 1- 10; - all these ranges being in mg xylanase protein per kg feed (ppm).
  • the xylanase is purified from the feed composition, and the specific activity of the purified xylanase is determined using a relevant assay (see under xylanase activity).
  • the xylanase activity of the feed composition as such is also determined using the same assay, and on the basis of these two determinations, the dosage in mg xylanase protein per kg feed is calculated.
  • the animal feed additive of the invention is intended for being included (orprescribed as having to be included) in animal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05 to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed). This is so in particular for premixes.
  • the present invention is also directed to methods for using the animal feed additive in animal feed.
  • animal includes all animals. Examples of animals are non-ruminants, and ruminants. Ruminant animals include, for example, animals such as sheep, goats, and cattle, e.g., beef cattle, cows, and young calves. In a particular embodiment, the animal is a nonruminant animal.
  • 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; and fish (including but not limited to salmon, trout, tilapia, catfish and carps; and crustaceans (including but not limited to shrimps and prawns).
  • 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; and fish (including but not limited to salmon, trout, tilapia, catfish
  • the animal feed additive can be fed to the animal before, after, or simultaneously with the diet.
  • the latter is preferred.
  • a well-defined additive preparation is advantageous. For instance, it is much easier to dose correctly to the feed a xylanase that is essentially free from interfering or contaminating other xylanases.
  • dose correctly refers in particular to the objective of obtaining consistent and constant results, and the capability of optimizing dosage based upon the desired effect.
  • the animal feed additive can be (a) added directly to the feed, or (b) it can be used in the production of one or more intermediate compositions such as premixes that are subsequently added to the feed (or used in a treatment process).
  • the present invention further relates to a method for improving the nutritional value of an animal feed comprising plant based material from the sub-family Panicoideae, comprising adding to the feed an animal feed additive.
  • the term improving the nutritional value of an animal feed means improving the availability of nutrients in the feed.
  • the nutritional values refer in particular to improving the solubilization and degradation of the arabinoxylan-containing fraction (e.g., such as hemicellulose) of the feed, thereby leading to increased release of nutrients from cells in the endosperm that have cell walls composed of highly recalcitrant hemicellulose. Consequently, an increased release of arabinoxylan oligomers indicates a disruption of the cell walls and as a result the nutritional value of the feed is improved resulting in increased growth rate and/or weight gain and/or feed conversion (i.e., the weight of ingested feed relative to weight gain).
  • arabinoxylan oligomer release may result in improved utilization of these components per se either directly or by bacterial fermentation in the hind gut thereby resulting in a production of short chain fatty acids that may be readily absorbed in the hind and utilised in the energy metabolism.
  • the invention further relates to a method of improving one or more performance parameters of an animal, comprising administering to one or more animals a animal feed additive and plant based material from the sub-family Panicoideae.
  • the plant based material from the sub-family Panicoideae may be administered together or separately with the animal feed additve.
  • the plant based material from the sub-family Panicoideae is maize, corn, sorghum, switchgrass, millet, pearl millet, foxtail millet or in a processed form such as milled corn, milled maize, defatted maize, defatted destarched maize, milled sorghum, milled switchgrass, milled millet, milled foxtail millet, milled pearl millet, or any combination thereof.
  • the plant based material from the subfamily Panicoideae is from the seed fraction (such as endosperm and/or husk) of the plant.
  • the improvement in the performance of an animal is an increase in body weight gain increases the Average Metabolizable Energy improved feed conversion ratio.
  • the improvement is an increased feed efficiency.
  • the improvement is an increase in body weight gain and/or an improved feed conversion ratio and/or an increased feed efficiency.
  • the improvement is the increase the Average Metabolizable Energy.
  • the invention further relates to methods of solubilizing xylan from a plant based material, e.g., by degrading the xylose backbone of sterically hindered arabinoxylan found in plant based material from the sub-family Panicoideae, thereby solubilizing increased amounts of arabinoxylan which is measured as arabinose and xylose.
  • Increased degradation, and thereby increased arabinose and xylose release can result in advantages for many industries which use plant based material from the sub-family Panicoideae.
  • model substrates wherein the starch and fat present in the plant material is removed without effecting the degree of substitution, can be used to aid the determination of improved enzyme combinations over known prior art combinations.
  • One model substrate is defatted destarched maize (DFDSM) and can be prepared as described in the Examples. It is important that the model substrate is not prepared using strongly acidic or basic conditions or high temperatures, since such conditions can remove the side chain carbohydrate molecules and/or ester groups present on the xylan backbone. If these side chain groups are removed, then the complexity and degree of substitution will be reduced resulting in an arabinoxylan material which is easy to degrade by known solutions. It is for this reason that heat, acid and/or base pre-treatment is used in biomass conversion.
  • the soluble arabinoxylan is hydrolyzed with acid resulting in xylose and arabinose being released into the supernatant. This xylose and arabinose is then detected using, e.g., the HPLC method as described herein.
  • the higher the degree of solubilization of the arabinoxylan the higher the amount of xylose and arabinose released upon acid hydrolysis. It is believed that increasing the solubilization of the arabinoxylan opens up the cell walls that can result in the nutrients, such as starch and protein, which are trapped inside being released. The release of starch and other nutrients can result in improved animal performance and/or improve the nutritional value of an animal feed.
  • the invention further relates to a method of releasing starch from plant based material, comprising treating plant based material from the sub-family Panicoideae with the enzyme combination.
  • the plant based material from the sub-family Panicoideae is from the tribe Andropogoneae such as the rank Andropogon or Andropterum or Apluda or Apocopis or Arthraxon or Bothriochloa or Capillipedium or Chionachne or Chrysopogon or Coelorachis or Coix or Cymbopogon or Dichanthium or Diheteropogon or Dimeria or Elionurus or Eremochloa or Euclasta or Eulalia or Germainia or Hemarthria or Heteropholis or Heteropogon or Hyparrhenia or Hyperthelia or Imperata or Ischaemum or Iseilema or Kerriochloa or Microstegium or Miscanthidium or Miscanthus or Mnesithea or Ophiuros or Oxyrhachis or Phacelurus or Pholiurus or Pogonatherum or Polytoca or Polytrias
  • sugar cane or Sarga or Schizachyrium or Sehima or Sorghastrum or Sorghum or Spodiopogon or Thaumastochloa or Thelepogon or Themeda or Trachypogon or Triarrhena or Tripsacum or Urelytrum or Vetiveria or Vossia or Xerochloa or Zea.
  • the plant based material from the sub-family Panicoideae is from the rank Zea, such as the species Zea diploperennis, Zea luxurians, Zea mays, Zea nicaraguensis or Zea perennis.
  • the plant based material from the sub-family Panicoideae is from the rank Sorghum, such as the species Sorghum amplum, Sorghum angustum, Sorghum arundinaceum, Sorghum australiense, Sorghum bicolor, Sorghum brachypodum, Sorghum bulbosum, Sorghum ecarinatum, Sorghum exstans, Sorghum grande, Sorghum halepense, Sorghum hybrid cultivar, Sorghum interjectum, Sorghum intrans, Sorghum laxiflorum, Sorghum leiocladum, Sorghum macrospermum, Sorghum matarankense, Sorghum nitidum, Sorghum plumosum, Sorghum propinquum, Sorghum purpureosericeum, Sorghum stipoideum, Sorghum sudanense, Sorghum timorense, Sorghum versicolor, Sorghum sp. 'Silk' or Sorghum
  • the plant based material from the sub-family Panicoideae is from the tribe Paniceae such as the rank Acritochaete, Acroceras, Alexfloydia, Alloteropsis, Amphicarpum, Ancistrachne, Anthephora, Brachiaria (e.g.
  • the plant based material from the sub-family Panicoideae is from the rank Panicum, such as the species Panicum adenophorum, Panicum aff. aquaticum JKT-2012, Panicum amarum, Panicum antidotale, Panicum aquaticum, Panicum arctum, Panicum arundinariae, Panicum atrosanguineum, Panicum auricomum, Panicum auritum, Panicum bartlettii, Panicum bergii, Panicum bisulcatum, Panicum boliviense, Panicum brazzavillense, Panicum brevifolium, Panicum caaguazuense, Panicum campestre, Panicum capillare, Panicum cayennense, Panicum cayoense, Panicum cervicatum, Panicum chloroleucum, Panicum claytonii, Panicum coloratum, Panicum cyanescen
  • the plant based material from the sub-family Panicoideae is maize (Zea), corn (Zea), sorghum (Sorghum), switchgrass ( Panicum virgatum), millet ( Panicum miliaceum), pearl millet ( Cenchrus violaceus also called Pennisetum glaucum), foxtail millet (Setaria italica also called Panicum italicum) or in a processed form such as milled corn, milled maize, defatted maize, defatted destarched maize, milled sorghum, milled switchgrass, milled millet, milled foxtail millet, milled pearl millet, or any combination thereof.
  • the plant based material from the sub-family Panicoideae is from the seed of the plant.
  • the plant based material from the sub-family Panicoideae is from the seed of maize (Zea), corn (Zea), sorghum (Sorghum), switchgrass (Panicum virgatum), millet (Panicum miliaceum), pearl millet (Cenchrus violaceus also called Pennisetum glaucum), foxtail millet (Setaria italica also called Panicum italicum) or wherein the seed has been processed such as milled corn, milled maize, defatted maize, defatted destarched maize, milled sorghum, milled switchgrass, milled millet, milled foxtail millet, milled pearl millet, or any combination thereof.
  • compositions described herein optionally include one or more additional enzymes.
  • Enzymes can be classified on the basis of the handbook Enzyme Nomenclature from NC-IUBMB, 1992), see also the ENZYME site at the internet: http://www.expasv.ch/enzvme/.
  • ENZYME is a repository of information relative to the nomenclature of enzymes. It is primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB), Academic Press, Inc., 1992, and it describes each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided (Bairoch A. The ENZYME database, 2000, Nucleic Acids Res 28:304-305). This IUB-MB Enzyme nomenclature is based on their substrate specificity and occasionally on their molecular mechanism; such a classification does not reflect the structural features of these enzymes.
  • glycoside hydrolase enzymes such as endoglucanase, galactanase, mannanase, dextranase, lysozyme and galactosidase is described in Henrissat et al, “The carbohydrate-active enzymes database (CAZy) in 2013”, Nucl. Acids Res. (1 January 2014) 42 (D1): D490-D495; see also www.cazy.org.
  • composition of the invention may also comprise at least one other enzyme selected from the group comprising of acetylxylan esterase (EC 3.1.1.23), acylglycerol lipase (EC).
  • acetylxylan esterase EC 3.1.1.23
  • acylglycerol lipase EC 3.1.1.23
  • composition of the invention comprises a galactanase (EC 3.2.1.89) and a beta-galactosidase (EC 3.2.1.23).
  • the composition of the invention comprises an alpha- galactosidase (EC 3.2.1.22).
  • the composition of the invention comprises a phytase (EC 3.1.3.8 or 3.1.3.26).
  • phytases include Bio-FeedTM Phytase (Novozymes), Ronozyme® P, Ronozyme® NP and Ronozyme® HiPhos (DSM Nutritional Products), NatuphosTM (BASF), NatuphosTM E (BASF), Finase® and Quantum® Blue (AB Enzymes), OptiPhos® (Huvepharma), AveMix® Phytase (Aveve Biochem), Phyzyme® XP (Verenium/DuPont) and Axtra® PHY (DuPont).
  • Other preferred phytases include those described in e.g. WO 98/28408, WO 00/43503, and WO 03/066847.
  • the composition of the invention comprises a xylanase (EC 3.2.1.8).
  • xylanases include Ronozyme® WX (DSM Nutritional Products), Econase® XT and Barley (AB Vista), Xylathin® (Verenium), Hostazym® X (Huvepharma), Axtra® XB (Xylanase/beta-glucanase, DuPont) and Axtra® XAP (Xylanase/amylase/protease, DuPont), AveMix® XG 10 (xylanase/glucanase) and AveMix® 02 CS (xylanase/glucanase/pectinase, Aveve Biochem), and Naturgrain (BASF).
  • the composition of the invention comprises a protease (EC 3.4).
  • protease EC 3.4
  • examples of commercially available proteases include Ronozyme® ProAct (DSM Nutritional Products).
  • the composition of the invention comprises an alpha-amylase (EC 3.2.1.1).
  • alpha-amylases include Ronozyme® A and RONOZYME® RumiStarTM (DSM Nutritional Products).
  • the composition of the invention comprises a multicomponent enzyme product, such as FRA® Octazyme (Framelco), Ronozyme® G2, Ronozyme® VP and Ronozyme® MultiGrain (DSM Nutritional Products), Rovabio® Excel or Rovabio® Advance (Adisseo).
  • a multicomponent enzyme product such as FRA® Octazyme (Framelco), Ronozyme® G2, Ronozyme® VP and Ronozyme® MultiGrain (DSM Nutritional Products), Rovabio® Excel or Rovabio® Advance (Adisseo).
  • Eubiotics are compounds which are designed to give a healthy balance of the micro-flora in the gastrointestinal tract. Eubiotics cover a number of different feed additives, such as probiotics, prebiotics, phytogenies (essential oils) and organic acids which are described in more detail below.
  • the animal feed composition further comprises one or more additional probiotic.
  • the animal feed composition further comprises a bacterium from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium and Megasphaera or any combination thereof.
  • animal feed composition further comprises a bacterium from one or more of the following strains: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Enterococcus faecium, Enterococcus spp, and Pediococcus spp, Lactobacillus spp, Bifidobacterium spp, Lactobacillus acidophilus, Pediococsus acidilactici, Lactococcus lactis, Bifidobacterium bifidum, Propionibacterium thoenii, Lactobacillus farciminus, lactobacillus rhamnosus, Clostridium butyricum, Bifidobacterium animalis ssp. animalis, Lactobacill
  • composition, animal feed additive or animal feed further comprises a bacterium from one or more of the following strains of Bacillus subtilis: 3A-P4 (PTA- 6506), 15A-P4 (PTA-6507), 22C-P1 (PTA-6508), 2084 (NRRL B-500130), LSSA01 (NRRL-B- 50104), BS27 (NRRL B-501 05), BS 18 (NRRL B-50633), BS 278 (NRRL B-50634), DSM 29870, DSM 29871 , DSM 32315, NRRL B-50136, NRRL B-50605, NRRL B-50606, NRRL B-50622 and PTA-7547.
  • a bacterium from one or more of the following strains of Bacillus subtilis: 3A-P4 (PTA- 6506), 15A-P4 (PTA-6507), 22C-P1 (PTA-6508), 2084 (NRRL B-500130), LSSA01 (NR
  • composition, animal feed additive or animal feed further comprises a bacterium from one or more of the following strains of Bacillus pumilus: NRRL B- 50016, ATCC 700385, NRRL B-50885 or NRRL B-50886.
  • composition, animal feed additive or animal feed further comprises a bacterium from one or more of the following strains of Bacillus lichenformis: NRRL B 50015, NRRL B-50621 or NRRL B-50623.
  • composition, animal feed additive or animal feed further comprises a bacterium from one or more of the following strains of Bacillus amyloliquefaciens: DSM 29869, DSM 29869, NRRL B 50607, PTA-7543, PTA-7549, NRRL B-50349, NRRL B- 50606, NRRL B-50013, NRRL B-50151, NRRL B-50141 , NRRL B-50147 or NRRL B-50888.
  • a bacterium from one or more of the following strains of Bacillus amyloliquefaciens: DSM 29869, DSM 29869, NRRL B 50607, PTA-7543, PTA-7549, NRRL B-50349, NRRL B- 50606, NRRL B-50013, NRRL B-50151, NRRL B-50141 , NRRL B-50147 or NRRL B-50888.
  • the bacterial count of each of the bacterial strains in the animal feed composition is between 1x10 4 and 1x10 14 CFU/kg of dry matter, preferably between 1x10 6 and 1x10 12 CFU/kg of dry matter, and more preferably between 1x10 7 and 1x10 11 CFU/kg of dry matter. In a more preferred embodiment the bacterial count of each of the bacterial strains in the animal feed composition is between 1x10 ® and 1x10 10 CFU/kg of dry matter.
  • the bacterial count of each of the bacterial strains in the animal feed composition is between 1x10 5 and 1x10 15 CFU/animal/day, preferably between 1x10 7 and 1x10 13 CFU/animal/day, and more preferably between 1x10 ® and 1x10 12 CFU/animal/day. In a more preferred embodiment the bacterial count of each of the bacterial strains in the animal feed composition is between 1x10 9 and 1x10 11 CFU/animal/day. In one embodiment, the amount of probiotics is 0.001% to 10% by weight of the composition.
  • the one or more bacterial strains are present in the form of a stable spore.
  • Examples of commercial products are Cylactin® (DSM Nutritional Products), Alterion (Adisseo), Enviva PRO (DuPont Animal Nutrition), Syncra® (mix enzyme + probiotic, DuPont Animal Nutrition), Ecobiol® and Fecinor® (Norel/Evonik) and GutCare® PY1 (Evonik).
  • Prebiotics are substances that induce the growth or activity of microorganisms (e.g., bacteria and fungi) that contribute to the well-being of their host.
  • Prebiotics are typically non- digestible fiber compounds that pass undigested through the upper part of the gastrointestinal tract and stimulate the growth or activity of advantageous bacteria that colonize the large bowel by acting as substrate for them.
  • prebiotics increase the number or activity of bifidobacteria and lactic acid bacteria in the Gl tract.
  • Yeast derivatives inactivated whole yeasts or yeast cell walls
  • prebiotics can also be considered as prebiotics. They often comprise mannan-oligosaccharids, yeast beta-glucans or protein contents and are normally derived from the cell wall of the yeast, Saccharomyces cerevisiae.
  • the amount of prebiotics is 0.001% to 10% by weight of the composition.
  • yeast products are Yang® and Agrimos (Lallemand Animal Nutrition).
  • Phytogenies are a group of natural growth promoters or non-antibiotic growth promoters used as feed additives, derived from herbs, spices or other plants.
  • Phytogenies can be single substances prepared from essential oils/extracts, essential oils/extracts, single plants and mixture of plants (herbal products) or mixture of essential oils/extracts/plants (specialized products).
  • phytogenies are rosemary, sage, oregano, thyme, clove, and lemongrass.
  • essential oils are thymol, eugenol, meta-cresol, vaniline, salicylate, resorcine, guajacol, gingerol, lavender oil, ionones, irone, eucalyptol, menthol, peppermint oil, alpha-pinene; limonene, anethol, linalool, methyl dihydrojasmonate, carvacrol, propionic acid/propionate, acetic acid/acetate, butyric acid/butyrate, rosemary oil, clove oil, geraniol, terpineol, citronellol, amyl and/or benzyl salicylate, cinnamaldehyde, plant polyphenol (tannin), turmeric and curcuma extract.
  • the amount of phytogeneics is 0.001% to 10% by weight of the composition.
  • Examples of commercial products are Crina® (DSM Nutritional Products); CinergyTM, BiacidTM, ProHacidTM Classic and ProHacidTM AdvanceTM (all Promivi/Cargill) and Envivo EO (DuPont Animal Nutrition).
  • Organic acids are widely distributed in nature as normal constituents of plants or animal tissues. They are also formed through microbial fermentation of carbohydrates mainly in the large intestine. They are often used in swine and poultry production as a replacement of antibiotic growth promoters since they have a preventive effect on the intestinal problems like necrotic enteritis in chickens and Escherichia coli infection in young pigs. Organic acids can be sold as mono component or mixtures of typically 2 or 3 different organic acids.
  • organic acids examples include propionic acid, formic acid, citric acid, lactic acid, sorbic acid, malic acid, acetic acid, fumaric acid, benzoic acid, butyric acid and tartaric acid or their salt (typically sodium or potassium salt such as potassium diformate or sodium butyrate).
  • the amount of organic acid is 0.001% to 10% by weight of the composition.
  • examples of commercial products are VevoVitall® (DSM Nutritional Products), Amasil®, Luprisil®, Lupro-Grain®, Lupro-Cid®, Lupro-Mix® (BASF), n-Butyric Acid AF (OXEA) and Adimix Precision (Nutriad).
  • a premix designates a preferably uniform mixture of one or more microingredients with diluent and/or carrier. Premixes are used to facilitate uniform dispersion of micro-ingredients in a larger mix.
  • a premix according to the invention can be added to feed ingredients or to the drinking water as solids (for example as water soluble powder) or liquids.
  • composition of the invention may further comprise one or more amino acids.
  • amino acids which are used in animal feed are lysine, alanine, beta-alanine, threonine, methionine and tryptophan.
  • the amount of amino acid is 0.001% to 10% by weight of the composition.
  • the animal feed may include one or more vitamins, such as one or more fat-soluble vitamins and/or one or more water-soluble vitamins.
  • the animal feed may optionally include one or more minerals, such as one or more trace minerals and/or one or more macro minerals.
  • fat- and water-soluble vitamins, as well as trace minerals form part of a so-called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed.
  • Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, e.g., vitamin K3.
  • Non-limiting examples of water-soluble vitamins include vitamin C, vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g., Ca-D- panthothenate.
  • Non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, iodine, selenium and zinc.
  • Non-limiting examples of macro minerals include calcium, magnesium, phosphorus, potassium and sodium.
  • the amount of vitamins is 0.001% to 10% by weight of the composition. In one embodiment, the amount of minerals is 0.001% to 10% by weight of the composition.
  • the animal feed additive of the invention comprises at least one of the individual components specified in Table A of WO 01/58275. At least one means either of, one or more of, one, or two, or three, or four and so forth up to all thirteen, or up to all fifteen individual components. More specifically, this at least one individual component is included in the additive of the invention in such an amount as to provide an in-feed-concentration within the range indicated in column four, or column five, or column six of Table A. In a still further embodiment, the animal feed additive of the invention comprises at least one of the below vitamins, preferably to provide an in-feed-concentration within the ranges specified in the below Table 1 (for piglet diets, and broiler diets, respectively).
  • composition of the invention may further comprise colouring agents, stabilisers, growth improving additives and aroma compounds/flavourings, polyunsaturated fatty acids (PUFAs); reactive oxygen generating species, antioxidants, anti-microbial peptides, anti-fungal polypeptides and mycotoxin management compounds.
  • colouring agents such as colouring agents, stabilisers, growth improving additives and aroma compounds/flavourings, polyunsaturated fatty acids (PUFAs); reactive oxygen generating species, antioxidants, anti-microbial peptides, anti-fungal polypeptides and mycotoxin management compounds.
  • PUFAs polyunsaturated fatty acids
  • colouring agents are carotenoids such as beta-carotene, astaxanthin, and lutein.
  • aroma compounds/flavourings are creosol, anethol, deca-, undeca-and/or dodeca-lactones, ionones, irone, gingerol, piperidine, propylidene phatalide, butylidene phatalide, capsaicin and tannin.
  • antimicrobial peptides examples include CAP18, Leucocin A, Tritrpticin, Protegrin- 1 , Thanatin, Defensin, Lactoferrin, Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000), Plectasins, and Statins, including the compounds and polypeptides disclosed in WO 03/044049 and WO 03/048148, as well as variants or fragments of the above that retain antimicrobial activity.
  • AFP antifungal polypeptides
  • Aspergillus giganteus and Aspergillus niger peptides, as well as variants and fragments thereof which retain antifungal activity, as disclosed in WO 94/01459 and WO 02/090384.
  • polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma- linoleic acid.
  • reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a syntethase.
  • Antioxidants can be used to limit the number of reactive oxygen species which can be generated such that the level of reactive oxygen species is in balance with antioxidants.
  • Mycotoxins such as deoxynivalenol, aflatoxin, zearalenone and fumonisin can be found in animal feed and can result in nmegative animal performance or illness.
  • mycotoxin management compounds are Vitafix®, Vitafix Ultra (Nuscience), Mycofix®, Mycofix® Secure, FUMzyme®, Biomin® BBSH, Biomin® MTV (Biomin), Mold-Nil®, Toxy-Nil® and Unike® Plus (Nutriad).
  • polypeptide having alpha- galactosidase activity is a GH36 alpha-galactosidase selected from the group consisting of a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide having at least 90% sequence identity to SEQ ID NO: 9; g) a polypeptide having at least 90% sequence identity to SEQ ID NO: 11 ; h) a polypeptide having at least 90% sequence identity to SEQ ID NO: 12; i) a polypeptide having at least 90% sequence identity to SEQ ID NO: 14; j)
  • L a polypeptide having at least 90% sequence identity to SEQ ID NO: 18; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; n) a polypeptide having at least 90% sequence identity to SEQ ID NO: 21 ; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO: 22.
  • polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; q) a polypeptide having at least 90% sequence identity to SEQ ID NO: 24; r) a polypeptide having at least 90% sequence identity to SEQ ID NO: 25; s) a polypeptide having at least 90% sequence identity to SEQ ID NO: 26; t) a polypeptide having at least 90% sequence identity to SEQ ID NO: 27; u) a polypeptide having at least 90% sequence identity to SEQ ID NO: 28; v) a polypeptide having at least 90% sequence identity to SEQ ID NO: 29; w) a polypeptide having at least 90% sequence identity to SEQ ID NO: 30; x) a polypeptide having at least 90% sequence identity to SEQ ID NO: 31 ; y) a polypeptide selected from the group consisting of p) a polypeptide
  • polypeptide having alpha-galactosidase activity is a GH36 alpha-galactosidase selected from the group consisting of a) a polypeptide obtained or obtainable from Bacillus deramificans having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide obtained or obtainable from Bacillus deramificans having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide obtained or obtainable from Bacillus acidopullulyticus having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide obtained or obtainable from Bacillus acidopullulyticus having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide obtained or obtainable from Anoxybacillus bogrovensis having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide obtained or obtainable from Anoxybacillus bo,
  • polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide obtained or obtainable from Bacillus subtilis having at least 90% sequence identity to SEQ ID NO: 23; q) a polypeptide obtained or obtainable from Bacillus amyloliquefaciens having at least 90% sequence identity to SEQ ID NO: 24; r) a polypeptide obtained or obtainable from Bacillus licheniformis having at least 90% sequence identity to SEQ ID NO: 25; s) a polypeptide obtained or obtainable Bacillus subtilis having at least 90% sequence identity to SEQ ID NO: 26; t) a polypeptide obtained or obtainable from Paenibacillus pabuli having at least 90% sequence identity to SEQ ID NO: 27; u) a polypeptide obtained or obtainable from Bacillus amyloliquefaciens having at least 90% sequence identity
  • polypeptide having alpha- galactosidase activity is a polypeptide selected from a polypetide as defined by a), b), c), d), e), f), k), m), and o).
  • polypeptide having alpha- galactosidase activity is a polypeptide selected from a polypetide as defined by k), m), and o).
  • polypeptide having alpha- galactosidase activity is a polypeptide selected from a polypetide as defined by k).
  • polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p), v) and gg).
  • An animal feed additive comprising a polypeptide having alpha-galactosidase activity and a polypeptide having xylanase activity; wherein the polypeptide having alpha-galactosidase activity is a GH36 alpha-galactosidase and wherein the polypeptide having xylanase activity is a GH30 xylanase.
  • polypeptide having alpha-galactosidase activity is a GH36 alpha-galactosidase selected from the group consisting of a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide having at least 90% sequence identity to SEQ ID NO: 9; g) a polypeptide having at least 90% sequence identity to SEQ ID NO: 11 ; h) a polypeptide having at least 90% sequence identity to SEQ ID NO: 12; i) a polypeptide having at least 90% sequence identity to SEQ ID NO: 14; j)
  • polypeptide having alpha-galactosidase activity is a polypeptide selected from the group consisting of a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide having at least 90% sequence identity to SEQ ID NO: 9; k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; n) a polypeptide having at least 90% sequence identity to SEQ ID NO: 21 ; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO:
  • polypeptide having alpha-galactosidase activity is a polypeptide selected from the group consisting of excellent) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO: 22 wherein the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; u) a polypeptide having at least 90% sequence identity to SEQ ID NO: 29; and ff) a polypeptide having at least 90% sequence identity to SEQ ID NO: 40.
  • the animal feed additive according to any of embodiments 11 to 15 further comprising one or more additional components selected from the group consisting of: one or more vitamins; one or more minerals; one or more amino acids; one or more phytogenies; one or more prebiotics; one or more organic acids; and one or more other feed ingredients
  • a method of improving the Average Metabolizable Energy of plant-based diet in a monogastric animal comprising administering an animal feed additive as defined in any of embodiments 11-16 or the combination as defined in any of embodiments 1 to 10.
  • composition comprising the combination as defined in any of embodiments 1 to 10.
  • composition according to embodiment 19 in the form of a granule.
  • composition according to embodiment 19 in the form of a liquid formulation.
  • An animal feed comprising the combination as defined in any of embodiments 1 to 10, or the animal feed additive as defined in any of embodiments 11 to 17, and further comprising plant-based material.
  • a method of releasing starch from plant-based material in animal feed comprising treating plant based material with the combination as defined in any of embodiments 1 to 10, or the animal feed additive as defined in any of embodiments 11 to 17 or the composition as defined in any of embodiments 19 to 21.
  • a method for improving the nutritional value of an animal feed, said feed comprising plant based material comprising adding to the feed comprising plant based material the combination as defined in any of embodiments 1 to 10, or the animal feed additive as defined in any of embodiments 11 to 17 or the composition as defined in any of embodiments 19 to 21.
  • the monogastric animal is selected from the group consisting of 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).
  • a method of feeding an animal comprising adding the combination as defined in any of embodiments 1 to 10, or the animal feed additive as defined in any of embodiments 11 to 17 or the composition as defined in any of embodiments 19 to 21 to the animal feed.
  • the animal feed according to embodiment 22 comprising maize, soybean meal or both.
  • the animal feed according to embodiment 22 comprising as a primary protein source maize or soybean meal or a combination thereof.
  • composition according to embodiment 19 and further comprising a formulating agent are provided.
  • composition of embodiment 30, wherein the formulating agent comprises one or more of the following compounds: 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, maltodextrin, cyclodextrin, wheat, PVA, acetate, phosphate and cellulose.
  • the formulating agent comprises one or more of the following compounds: 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
  • composition according to embodiment 19 further comprising one or more additional enzymes.
  • composition of embodiment 32, wherein the one or more additional enzymes is selected from the group consisting of acetyl xylan esterase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha- galactosidase, beta-galactosidase, beta-glucanase, beta-glucosidase, lipase, glucan 1 ,4- a-glucosidase, glucan 1 ,4-alpha-maltohydrolase, lysophospholipase, lysozyme, mannanase, alpha-mannosidase, beta-mannosidase, phytase, phospholipase A1, phospholipase A2, phospholipase C, phospholipase D, protease, pullulan
  • composition of any of embodiments 30 to 35, further comprising plant based material is provided.
  • composition of embodiment 36, wherein the plant based material is from the sub-family Panicoideae.
  • composition of embodiment 37, wherein the plant based material from the sub-family Panicoideae is maize, corn, sorghum, switchgrass, millet, pearl millet, foxtail millet or in a processed form such as milled corn, milled maize, defatted maize, defatted destarched maize, milled sorghum, milled switchgrass, milled millet, milled foxtail millet, milled pearl millet, or any combination thereof.
  • composition of embodiments 36 to 38, wherein the plant based material from the subfamily Panicoideae is from the seed fraction (such as endosperm and/or husk) of the plant.
  • a granule comprising the combination of any of embodiments 1 to 10 and a formulating agent.
  • the granule of embodiment 40 wherein the one or more formulating agents is 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, preferably selected from the list consisting of 1 , 2-propylene glycol, 1 , 3-propylene glycol, sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate. 42. The granule of any of embodiments 40 to 41 , wherein the granule comprises a core particle and one or more coatings
  • Cage broiler studies are designed for the evaluation of the combination of soy carbohydrate degrading enzymes on growth performance and apparent metabolisable energy corrected to zero N retention.
  • the present describes the mandatory conditions/specifications for each internal and external trial.
  • A NC; B: triple high - comprising SEQ ID NO:16 - 20ppm EP each : C: triple low - comprising SEQ ID NO:16 - 5ppm EP each; D: single high - comprising SEQ ID NO:16 - 20ppm EP; E: single low - comprising SEQ ID NO:16 - 5ppm EP
  • Feed distribution pellets or mash, according to the institute selected and available
  • Final volume of the product solution is expected to be: 300 to 500 ml for ⁇ 200 kg diet.
  • indigestible marker should be included in the experimental diets (i.e: Titanium dioxide T1O2, acid-insoluble ash or chromium oxide). Alternatively a total collection method may be used.
  • Nitrogen is determined using automated nitrogen analyser (LECO) according to Dumas combustion method or using Kjeldahl procedure (protein contents are calculated using the formula: 6.25 x N).
  • Gross energy is determined using an adiabatic bomb calorimeter standardized with benzoic acid.
  • N-corrected apparent metabolizable energy (AMEn) values are calculated according to the following equation:
  • a total collection method may be used or, after sampling marker analysis may be conducted as per the institutes protocols.
  • the granule is prepared by granulating a xylanase variant of the invention with a filler such as sodium sulfate, magnesium sulfate, calcium carbonate and/or cellulose and then optionally coating the granule with a wax coating (e.g. hydrogenated palm oil) or a salt coating (e. g. sodium sulfate and/or magnesium sulfate).
  • a filler such as sodium sulfate, magnesium sulfate, calcium carbonate and/or cellulose
  • a salt coating e. g. sodium sulfate and/or magnesium sulfate
  • granule is prepared by absorbing a liquid solution of a xylanase variant of the invention onto an inert core and then optionally coating the granule with a wax coating (e.g. hydrogenated palm oil) or a salt coating (e. g. sodium sulfate and/or magnesium sulfate).
  • a wax coating e.g. hydrogenated palm oil
  • a salt coating e. g. sodium sulfate and/or magnesium sulfate
  • premix formulation of a xylanase variant of the invention containing 0.01 g to 10g enzyme protein per kilo of premix is added to the following premix:
  • Vitamin B1 2500 mg Vitamin B2
  • animal feed comprising the animal feed additive as described above:
  • the ingredients are mixed, and the feed is pelleted at the desired temperature, e.g. 60, 65, 75, 80, 85, 90 or even 95°C.
  • a liquid formulation of a xylanase variant of the invention comprises 0.1% to 10 w/w enzyme protein, 40-60% glycerol, 0.1 to 0.5% sodium benzoate and water.
  • the liquid formulation is sprayed onto the pelleted animal feed described above.

Abstract

A combination of alpha-galactosidases and xylanases in animal feed increases the availability of the gross energy (GE) and the Average Metabolizable Energy of the animal feed by hydrolyzing the complex carbohydrates of maize, soybean meal and other protein meal sources. The hydrolysis of the indigestible oligosaccharides by the combination results also in reducing their anti-nutritional effect.

Description

ANIMAL FEED COMPOSITION
Reference to a Sequence Listing
This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.
Background of the Invention
Field of the Invention
The present invention relates to a combination of a xylanase and an alpha-galactosidase in an animal feed for improving the nutritional value of the animal feed.
Description of the Related Art
Xylans are hemicelluloses found in all land plants (Popper and Tuohy, 2010, Plant Physiology 153: 373-383). They are especially abundant in secondary cell walls and xylem cells. In grasses, with type II cell walls, glucurono arabinoxylans are the main hemicellulose and are present as soluble or insoluble dietary fiber in many grass based food and feed products.
Plant xylans have a b-1 ,4-linked xylopyranose backbone that can be substituted at the 02 or 03 position with arabinose, glucuronic acid and acetic acid in a species and tissue specific manner. The starch-rich seeds of the sub-family Panicoideae with economically important species such as corn, sorghum, rice and millet have special types of highly substituted xylans in their cell walls. Compared to wheat flour, wherein over 60% of the xylosyl units in the arabinoxylan backbone are unsubstituted. In corn kernel xylan, the corresponding percentage of unsubstituted backbone xylosyls is 20-30%, and in sorghum it is 35-40% (Huismann etal., 2000, Carbohydrate Polymers 42: 269-279). Furthermore, in corn and sorghum the xylan side chains can be longer than a single arabinose or glucuronic acid substitution which is common in other xylans. This added side chain complexity is often due to L- and D-galactose and D-xylose sugars bound to the side chain arabinose or glucuronic acid. About every tenth arabinose in corn kernel xylan is also esterified with a ferulic acid and about every fourth xylose carries an acetylation (Agger et a!., 2010, J. Agric. Food Chem. 58: 6141-6148). All of these factors combined make the highly substituted xylans in corn and sorghum resistant to degradation by traditional xylanases.
The known enzymes responsible for the hydrolysis of the xylan backbone are classified into enzyme families based on sequence similarity (cazy.org). The enzymes with mainly endo- xylanase activity have previously been described in Glycoside hydrolase family (GH) 5, 8, 10, 11 , 30 and 98. The enzymes within a family share some characteristics such as 3D fold and they usually share the same reaction mechanism. Some GH families have narrow or mono-specific substrate specificities while other families have broad substrate specificities.
Commercially available GH10 and GH11 xylanases are often used to break down the xylose backbone of arabinoxylan. In animal feed this results in a degradation of the cereal cell wall with a subsequent improvement in nutrient release (starch and protein) encapsulated within the cells. Degradation of xylan also results in the formation of xylose oligomers that may be utilised for hind gut fermentation and therefore can help an animal to obtain more digestible energy. However, such xylanases are sensitive to side chain steric hindrance and whilst they are effective at degrading arabinoxylan from wheat, they are not very effective on the xylan found in the seeds of Poaceae species, such as corn or sorghum.
Soybean meal is the second biggest crop globally & the biggest protein source. Soybean meal has high protein, low fat, low starch, and high NSP content. The gross energy (GE) in soybean meal (SBM) is approximately 17 MJ / kg (4000 kcal / kg). However, poultry utilizes only approximately 55%. Most of this unused energy consists of indigestible oligosaccharides and non-starch polysaccharides. The indigestible soluble oligosaccharides (raffinose, stachyose, verbascose) increase passage rates and inhibits the digestion and absorption of nutrients. Oligosaccharides are also fermented in the hind-gut, causing bloating, gas production and resulting in reductions in feed intake.
For the raffinose series oligosaccharides, the galactose monosaccharides are connected in a- 1.6 linkages between the galactose monomers and between galactose and sucrose.
Alpha-galactosidases have been selected and novel a-galactosidase have been developed to hydrolyze the complex carbohydrates of SBM and other protein meal sources, such as rapeseed, lupin, beans etc, to galactose and sucrose to increase the energy delivery from soybean meal and other feed sources. Alpha-galactosidases, together liberate an increased level of galactose and sucrose can be used by the animals as energy source. The hydrolysis of the indigestible oligosaccharides results also in reducing their anti-nutritional effect.
Rovabio® Advance comprises arabinofuranosidases (ABFs), and a complex mixture of different xylanases. This large range of xylanases in combination with ABFs explains the consistency of the results regardless of the feed composition.
US 6,399,123 claims a method of removing oligosaccharides from feed comprising adding galactosidase, xylanase and/or cellulase to the feed in an amount of 0.1% to 1.0% by weight of the feed, hydrolyzing the oligosaccharides and then consumption by an animal. US 6,399,123 further claims a method of improving BWG in chickens comprising adding a galactosidase to the feed to cause hydrolysis of the oligosaccharides and then feeding this treated feed to chickens.
WO 94/23022 discloses the use of a preferred alpha-galactosidase in animal feed.
Alpha-galactosidase is a glycoside hydrolase enzyme that hydrolyses the terminal alpha- galactosyl moieties from glycolipids and glycoproteins that is present in, e.g. legumes, vegetables, grains, cereals and the like. Alpha-galactosidases are produced by various microorganisms, plants and animals, but mammals are deficient in intestinal alpha-galactosidase production and consequently are incapable of decomposing ingested alpha-galactosides by themselves. Instead, ingested alpha-galactosides are decomposed by microorganisms present in the intestine.
Soybean is a species of legume native to East Asia and is the second biggest feed crop globally and the biggest protein source applied in animal feed. Soybean can be manufactured (defatted) to produce soybean meal (SBM), and SBM is a significant and cheap source of high quality protein for animal feeds. Other common types of legume are chickpea, lupin, lentil, peanut, beans or peas which can also be processed and used as animal feed. Legumes, such as soybean, contain significant amounts of raffinose oligosaccharides which requires an alpha- galactosidase present to release galactose.
Up to 70% of a farmer’s expenses is from the cost of animal feed. Thus, there is always an interest by farmers in either reducing feed costs by feeding less, or in obtaining improved animal growth using the same amount of feed. One way of achieving this is to release as much energy from the feed as possible by e.g. using enzymes such as alpha-galactosidases. However, enzymes are also an expense and therefore the alpha-galactosidase should work at a cost- effective dose.
GH36 alpha-galactosidase are known in the prior art, such as disclosed in W02009/108941 (SEQ ID NO: 597, AXR38459).
Summary of the Invention
Alpha-galactosidases and xylanases in animal feed increase the availability of the gross energy (GE) and the Average Metabolizable Energy of the animal feed by hydrolyzing the complex carbohydrates of maize, soybean meal and other protein meal sources. The hydrolysis of the indigestible oligosaccharides by the combination results also in reducing their anti- nutritional effect.
One aspect of the invention is directed to a use of a combination of a polypeptide having alpha-galactosidase activity and a polypeptide having xylanase activity in an animal feed or animal feed additive, wherein the polypeptide having alpha-galactosidase activity is a GH36 alpha-galactosidase and wherein the polypeptide having xylanase activity is a GH30 xylanase. A related aspect is directed to a composition comprising said combination. Similarly, the invention is directed to an animal feed additive comprising a polypeptide having alpha-galactosidase activity and a polypeptide having xylanase activity; wherein the polypeptide having alpha- galactosidase activity is a GH36 alpha-galactosidase and wherein the polypeptide having xylanase activity is a GH30 xylanase. A further aspect of the invention is directed to a method of improving the Average Metabolizable Energy of plant-based diet in a monogastric animal comprising administering an animal feed additive, composition or the combination of the invention.
A further aspect is directed to an animal feed comprising the combination, the animal feed additive, or the composition of the invention, and further comprising plant-based material.
A further aspect is directed to a method of releasing starch from plant-based material in animal feed, comprising treating plant-based material with the combination, the animal feed additive, or the composition of the invention. A further aspect is directed to a method for improving the nutritional value of an animal feed, said feed comprising plant-based material comprising adding to the feed comprising plant-based material and the combination, the animal feed additive, or the composition of the invention. A further aspect is directed to a method of feeding an animal comprising adding the combination, the animal feed additive, or the composition of the invention to an animal feed.
Brief Description of the Sequence Listing
SEQ ID NO: 1 is the gene sequence of GH36 alpha-galactosidase as isolated from Bacillus deramificans.
SEQ ID NO: 2 is the amino acid sequence as deduced from SEQ ID NO: 1.
SEQ ID NO: 3 is the amino acid sequence of SEQ ID NO: 2 with His-tag.
SEQ ID NO: 4 is the gene sequence of GH36 alpha-galactosidase as isolated from Bacillus acidopullulyticus.
SEQ ID NO: 5 is the amino acid sequence as deduced from SEQ ID NO: 4.
SEQ ID NO: 6 is the amino acid sequence of SEQ ID NO: 5 with His-tag.
SEQ ID NO: 7 is the gene sequence of GH36 alpha-galactosidase as isolated from Anoxybacillus bogrovensis.
SEQ ID NO: 8 is the amino acid sequence as deduced from SEQ ID NO: 7.
SEQ ID NO: 9 is the amino acid sequence of SEQ ID NO: 8 with His-tag.
SEQ ID NO: 10 is the gene sequence of GH36 alpha-galactosidase as isolated from Aspergillus sydowii.
SEQ ID NO: 11 is the amino acid sequence as deduced from SEQ ID NO: 10.
SEQ ID NO: 12 is the amino acid sequence of the mature GH36 alpha-galactosidase from Aspergillus sydowii.
SEQ ID NO: 13 is the cDNA sequence of GH36 alpha-galactosidase as isolated from Bacillus sp-19140.
SEQ ID NO: 14 is the amino acid sequence as deduced from SEQ ID NO: 13.
SEQ ID NO: 15 is the amino acid sequence of SEQ ID NO: 14 with His-tag. SEQ ID NO: 16 is the corrected amino acid sequence of the alpha-galactosidase as disclosed in W01994/23022.
SEQ ID NO: 17 is the cDNA sequence of GH36 alpha-galactosidase as isolated from Aspergillus puniceus.
SEQ ID NO: 18 is the amino acid sequence as deduced from SEQ ID NO: 17.
SEQ ID NO: 19 is the amino acid sequence of the mature GH36 alpha-galactosidase from Aspergillus puniceus.
SEQ ID NO: 20 is the cDNA sequence of GH36 alpha-galactosidase as isolated from Penicillium pseudopulvillorum .
SEQ ID NO: 21 is the amino acid sequence as deduced from SEQ ID NO: 20.
SEQ ID NO: 22 is the amino acid sequence of the mature GH36 alpha-galactosidase from Penicillium pseudopulvillorum .
SEQ ID NO: 23 is the amino acid sequence of a mature GH30 xylanase from Bacillus subtilis.
SEQ ID NO: 24 is the amino acid sequence of a mature GH30 xylanase from Bacillus amyloliquefaciens.
SEQ ID NO: 25 is the amino acid sequence of a mature GH30 xylanase from Bacillus Hcheniformis.
SEQ ID NO: 26 is the amino acid sequence of a mature GH30 xylanase from Bacillus subtilis.
SEQ ID NO: 27 is the amino acid sequence of a mature GH30 xylanase from Paenibacillus pabuli.
SEQ ID NO: 28 is the amino acid sequence of a mature GH30 xylanase from Bacillus amyloliquefaciens HB-26.
SEQ ID NO: 29 is the amino acid sequence of a mature GH30 xylanase (as defined in WO03106654 by SEQ ID NO: 190).
SEQ ID NO: 30 is the amino acid sequence of the mature GH30_8 xylanase from Clostridium acetobutylicum:
SEQ ID NO: 31 is the amino acid sequence of the mature GH30_8 xylanase from Pseudoalteromonas tetraodonis.
SEQ ID NO: 32 is the amino acid sequence of the mature GH30_8 xylanase from Paenibacillus sp-19179.
SEQ ID NO: 33 is the amino acid sequence of the mature GH30_8 xylanase Pectobacterium carotovorum subsp. Carotovorum:
SEQ ID NO: 34 is the amino acid sequence of the mature GH30_8 xylanase Ruminococcus sp. CAG:330. SEQ ID NO: 35 is the amino acid sequence of the mature GH30_8 xylanase Streptomyces sp-62627.
SEQ ID NO: 36 is the amino acid sequence of the mature GH30_8 xylanase Clostridium saccharoperb utylacetonicum .
SEQ ID NO: 37 is the amino acid sequence of the mature GH30_8 xylanase Paenibacillus panacisoli.
SEQ ID NO: 38 is the amino acid sequence of the mature GH30_8 xylanase Human Stool metagenome.
SEQ ID NO:39 is the amino acid sequence of the mature GH30_8 xylanase Vibrio rhizosphaerae .
SEQ ID NO:40 is the amino acid sequence of the mature GH30_8 xylanase from Bacillus sp-18423.
Detailed Description of the Invention
Aspects of the invention include the use of a combination of a polypeptide having alpha- galactosidase activity and a polypeptide having xylanase activity in an animal feed or animal feed additive, wherein the polypeptide having alpha-galactosidase activity is a GH36 alpha- galactosidase and wherein the polypeptide having xylanase activity is a GH30 xylanase.
Definitions
Xylanase: The term “xylanase” means a glucuronoarabinoxylan endo-1 ,4-beta-xylanase (E.C. 3.2.1.136) that catalyses the endohydrolysis of 1 ,4-beta-D-xylosyl links in some glucuronoarabinoxylans. Xylanase activity can be determined with 0.2% AZCL-glucuronoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C. One unit of xylanase activity is defined as 1 .0 pmole of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-glucuronoxylan as substrate in 200 mM sodium phosphate pH 6.
Alpha-galactosidase: The term “alpha-galactosidase”, also called a-D-galactoside galactohydrolase (E.C. 3.2.1.22), means an enzyme that catalyses the hydrolysis of terminal, non-reducing a-D-galactose residues in a-D-galactosides, such as galactose oligosaccharides, galactomannans and galactolipids. Alpha-galactosidase activity can be determined using 4- nitrophenyl a-D-galactopyranoside (available from Megazyme International, Bray, Co. Wicklow, Ireland) as substrate in 100 mM MES (Sigma) buffer pH 7.0 ± 0.05 at room temperature. The enzyme is diluted in 2-fold dilutions and then the 4-nitrophenyl a-D-galactopyranoside substrate is dissolved in the solution containing the enzyme. The alpha-galactosidase activity is followed directly in the buffer by measuring the absorbance of released pNP at 405 nm as function of time. A detailed assay can be found in the alpha-galactosidase assay as described herein. Allelic variant: The term “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.
Animal: The term “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, pompano, roach, salmon, sampa, sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleye and whitefish); and crustaceans (including but not limited to shrimps and prawns).
Animal feed: The term “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).
Arabinoxylan-containing material: The term “Arabinoxylan-containing material” means any material containing arabinoxylan. Arabinoxylan is a hemicellulose found in both the primary and secondary cell walls of plants, including woods and cereal grains, consisting of copolymers of two pentose sugars, arabinose and xylose. The arabinoxylan chain contains a large number of 1 ,4-linked xylose units. Many xylose units are substituted with 2-, 3- or 2,3-substituted arabinose residues.
Examples of arabinoxylan-containing material are forage, roughage, seeds and grains (either whole or prepared by crushing, milling, etc from, e.g., corn, oats, rye, barley, wheat), trees or hard woods (such as poplar, willow, eucalyptus, palm, maple, birch), bamboo, herbaceous and/or woody energy crops, agricultural food and feed crops, animal feed products, cassava peels, cocoa pods, sugar cane, sugar beet, locust bean pulp, vegetable or fruit pomaces, wood waste, bark, shavings, sawdust, wood pulp, pulping liquor, waste paper, cardboard, construction and demolition wood waste, industrial or municipal waste water solids or sludge, manure, by product from brewing and/or fermentation processes, wet distillers grain, dried distillers grain, spent grain, vinasse and bagasse.
Forage as defined herein also includes roughage. Forage is fresh plant material such as hay and silage from forage plants, grass and other forage plants, grass and other forage plants, seaweed, sprouted grains and legumes, or any combination thereof. Examples of forage plants are Alfalfa (Lucerne), birdsfoot trefoil, brassica (e.g., kale, rapeseed (canola), rutabaga (swede), turnip), clover (e.g., alsike clover, red clover, subterranean clover, white clover), grass (e.g., Bermuda grass, brome, false oat grass, fescue, heath grass, meadow grasses, miscanthus, orchard grass, ryegrass, switchgrass, Timothy-grass), corn (maize), hemp, millet, barley, oats, rye, sorghum, soybeans and wheat and vegetables such as beets. Crops suitable for ensilage are the ordinary grasses, clovers, alfalfa, vetches, oats, rye and maize. Forage further includes crop residues from grain production (such as corn stover; straw from wheat, barley, oat, rye and other grains); residues from vegetables like beet tops; residues from oilseed production like stems and leaves form soy beans, rapeseed and other legumes; and fractions from the refining of grains for animal or human consumption or from fuel production or other industries.
Roughage is generally dry plant material with high levels of fiber, such as fiber, bran, husks from seeds and grains and crop residues (such as stover, copra, straw, chaff, sugar beet waste).
Preferred sources of arabinoxylan-containing materials are forage, roughage, seeds and grains, sugar cane, sugar beet and wood pulp.
Body Weight Gain: The term “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: The term “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.
Coding sequence: The term “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: The term “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 (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the variant or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, 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.
Expression: The term “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: The term “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.
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. By "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: The term “feed efficiency” means the amount of weight gain per unit of feed when the animal is fed ad-libitum or a specified amount of feed during a period of time. By "increased 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: The term “fragment” means a polypeptide having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide; wherein the fragment has xylanase activity. In one aspect, a fragment comprises at least 330 amino acid residues, at least 350 amino acid residues, or at least 370 amino acid residues.
Highly branched xylan: The term “highly branched xylan” means that more than 50% of xylosyl units in the arabinoxylan backbone are substituted. This is preferably calculated from linkage analysis as performed in Huismann et al. Carbohydrate Polymers, 2000, 42:269-279.
Host cell: The term “host cell” means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. 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. Improved property: The term “improved property” means a characteristic associated with a variant that is improved compared to a parent. Such improved properties include, but are not limited to, catalytic efficiency, catalytic rate, chemical stability, oxidation stability, pH activity, pH stability, specific activity, stability under storage conditions, substrate binding, substrate cleavage, substrate specificity, substrate stability, surface properties, thermal activity, and thermostability. In an embodiment, the improved property is improved thermostability.
Isolated: The term “isolated” means a substance in a form or environment which does not occur in nature. Non-limiting examples of 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: The term “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.
It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.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: The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having xylanase activity.
Mutant: The term “mutant” means a polynucleotide encoding a variant.
Nucleic acid construct: The term "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: The term “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 as used herein 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 digesta on a specified segment of the gastro-intestinal tract of the animal, e.g., the ileum. Metabolizable energy as used herein 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 digesta 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.
Operably linked: The term “operably 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: The term “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. For the purpose of the present invention, 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 (DFDSM) 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. Thus 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. In a more detailed embodiment, 2% (w/w) DFDSM suspension was prepared in 100 mM sodium acetate, 5 mM CaCI , pH 5 and allowed to hydrate for 30 min at room temperature under gently stirring. After hydration, 200 pi substrate suspension was 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). The 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 mI supernatant was mixed with 100 mI 1.6 M HCI and transferred to 300 mI PCR tubes and left for acid hydrolysis for 40 min at 90°C in a PCR machine. Samples were neutralized with 125 mI 1.4 M NaOH after acid hydrolysis and loaded on the HPAE-PAD for mono-saccharide analysis.
Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.
For purposes of the present invention, the 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, 2000, Trends Genet. 16: 276-277), 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 EBLOSUM62 (EMBOSS version of BLOSUM62) 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:
(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)
For purposes of the present invention, the sequence identity between two 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:
(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)
Subsequence: The term “subsequence” means a polynucleotide having one or more (e.g., several) nucleotides absent from the 5' and/or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having xylanase activity.
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 + Ser411Phe” or “G205R + S411F”, 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, *. Accordingly, the 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”.
In such cases 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). In the above example, the sequence would thus be:
Figure imgf000014_0001
Multiple alterations. 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.
Different alterations. Where different alterations can be introduced at a position, the different alterations are separated by a comma, e.g., “Arg170Tyr,Glu” represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala + Arg170Gly,Ala” designates the following variants:
“Tyr 167G ly+ Arg 170G ly” , “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and
“Tyr167Ala+Arg170Ala”.
Xylanases of the Invention
The polypeptide having xylanase activity are xylanases of the GH30 family, preferably GH30 subfamily 8 variants. In a suitable embodiment, the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; q) a polypeptide having at least 90% sequence identity to SEQ ID NO: 24; r) a polypeptide having at least 90% sequence identity to SEQ ID NO: 25; s) a polypeptide having at least 90% sequence identity to SEQ ID NO: 26; t) a polypeptide having at least 90% sequence identity to SEQ ID NO: 27; u) a polypeptide having at least 90% sequence identity to SEQ ID NO: 28; v) a polypeptide having at least 90% sequence identity to SEQ ID NO: 29; w) a polypeptide having at least 90% sequence identity to SEQ ID NO: 30; x) a polypeptide having at least 90% sequence identity to SEQ ID NO: 31 ; y) a polypeptide having at least 90% sequence identity to SEQ ID NO: 32; z) a polypeptide having at least 90% sequence identity to SEQ ID NO: 33; aa) a polypeptide having at least 90% sequence identity to SEQ ID NO: 34; bb) a polypeptide having at least 90% sequence identity to SEQ ID NO: 35; cc) a polypeptide having at least 90% sequence identity to SEQ ID NO: 36; dd) a polypeptide having at least 90% sequence identity to SEQ ID NO: 37; ee) a polypeptide having at least 90% sequence identity to SEQ ID NO: 38; ft) a polypeptide having at least 90% sequence identity to SEQ ID NO: 39; and gg) a polypeptide having at least 90% sequence identity to SEQ ID NO: 40.
In a futher suitable embodiment, the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide obtained or obtainable from Bacillus subtilis having at least 90% sequence identity to SEQ ID NO: 23; q) a polypeptide obtained or obtainable from Bacillus amyloliquefaciens having at least 90% sequence identity to SEQ ID NO: 24; r) a polypeptide obtained or obtainable from Bacillus Hcheniformis having at least 90% sequence identity to SEQ ID NO: 25; s) a polypeptide obtained or obtainable Bacillus subtilis having at least 90% sequence identity to SEQ ID NO: 26; t) a polypeptide obtained or obtainable from Paenibacillus pabuli having at least 90% sequence identity to SEQ ID NO: 27; u) a polypeptide obtained or obtainable from Bacillus amyloliquefaciens having at least 90% sequence identity to SEQ ID NO: 28; v) a polypeptide obtained or obtainable from Bacillus subtilis having at least 90% sequence identity to SEQ ID NO: 29; w) a polypeptide obtained or obtainable from Clostridium acetobutylicum having at least 90% sequence identity to SEQ ID NO: 30; x) a polypeptide obtained or obtainable from Pseudoalteromonas tetraodonis having at least 90% sequence identity to SEQ ID NO: 31 ; y) a polypeptide obtained or obtainable from Paenibacillus sp-19179 having at least 90% sequence identity to SEQ ID NO: 32; z) a polypeptide obtained or obtainable from Pectobacterium carotovorum having at least 90% sequence identity to SEQ ID NO: 33; aa) a polypeptide obtained or obtainable from Ruminococcus sp. having at least 90% sequence identity to SEQ ID NO: 34; bb) a polypeptide obtained or obtainable from Streptomyces sp-62627 having at least 90% sequence identity to SEQ ID NO: 35; cc) a polypeptide obtained or obtainable from Clostridium saccharoperbutylacetonicum having at least 90% sequence identity to SEQ ID NO: 36; dd) a polypeptide obtained or obtainable from Paenibacillus panacisoli having at least 90% sequence identity to SEQ ID NO: 37; ee) a polypeptide obtained or obtainable from Human Stool metagenome having at least 90% sequence identity to SEQ ID NO: 38; ff) a polypeptide obtained or obtainable from Vibrio rhizosphaerae having at least 90% sequence identity to SEQ ID NO: 39; and gg) a polypeptide obtained or obtainable from Bacillus sp-18423 having at least 90% sequence identity to SEQ ID NO: 40.
In a preferred embodiment, the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; u) a polypeptide having at least 90% sequence identity to SEQ ID NO: 29; and gg) a polypeptide having at least 90% sequence identity to SEQ ID NO: 40. More preferably, the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; and gg) a polypeptide having at least 90% sequence identity to SEQ ID NO: 40, such as wherein polypeptide having xylanase activity is a GH30 xylanase having at least 90% sequence identity to SEQ ID NO: 23.
The xylanase of the combination of the invention may be, in one embodiment , selected from SEQ ID NO: 29, and comprising a substitution at one or more (e.g., several) positions corresponding to positions 24, 26, 36, 37, 60, 71 , 74, 75, 76, 124, 133, 155, 167, 208, 317, and 321 of SEQ ID NO: 29, wherein the variant has xylanase activity and wherein the variant has xylanase activity and has at least 70% identity, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, or most preferably at least 95% identity to SEQ ID NO: 29.
In another embodiment, the polypeptide having xylanase activity has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 23.
In another embodiment, the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 24.
In another embodiment, the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 25.
In another embodiment, the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 26.
In another embodiment, the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 27.
In another embodiment, the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 28.
In another embodiment, the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 29.
In another embodiment, the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO:30.
In another embodiment, the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 31.
In another embodiment, the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 32.
In another embodiment, the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 33.
In another embodiment, the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 34.
In another embodiment, the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 35.
In another embodiment, the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 36.
In another embodiment, the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 37.
In another embodiment, the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 38.
In another embodiment, the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 39.
In another embodiment, the xylanase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 40.
In another embodiment, the xylanase comprises or consists of the substitutions H24W + A26E of the polypeptide of SEQ ID NO: 29. In another embodiment, the xylanase comprises or consists of the substitutions H24W + V74L of the polypeptide of SEQ ID NO: 29.
In another embodiment, the xylanase comprises or consists of the substitutions H24W + K75L of the polypeptide of SEQ ID NO: 29.
In another embodiment, the xylanase comprises or consists of the substitutions H24W + H76L of the polypeptide of SEQ ID NO: 29.
In another embodiment, the xylanase comprises or consists of the substitutions H24W + I155M of the polypeptide of SEQ ID NO: 29.
In another embodiment, the xylanase comprises or consists of the substitutions H24W + V208L of the polypeptide of SEQ ID NO: 29.
In another embodiment, the xylanase comprises or consists of the substitutions A26E + V74L of the polypeptide of SEQ ID NO: 29.
In another embodiment, the xylanase comprises or consists of the substitutions A26E + K75L of the polypeptide of SEQ ID NO: 29.
In another embodiment, the xylanase comprises or consists of the substitutions A26E + H76L of the polypeptide of SEQ ID NO: 29.
In another embodiment, the xylanase comprises or consists of the substitutions A26E + I155M of the polypeptide of SEQ ID NO: 29.
In another embodiment, the xylanase comprises or consists of the substitutions A26E + V208L of the polypeptide of SEQ ID NO: 29.
In another embodiment, the xylanase comprises or consists of the substitutions V74L + K75L of the polypeptide of SEQ ID NO: 29.
In another embodiment, the xylanase comprises or consists of the substitutions V74L + H76L of the polypeptide of SEQ ID NO: 29.
In another embodiment, the xylanase comprises or consists of the substitutions V74L + I155M of the polypeptide of SEQ ID NO: 29.
In another embodiment, the xylanase comprises or consists of the substitutions V74L + V208L of the polypeptide of SEQ ID NO: 29.
In another embodiment, the xylanase comprises or consists of the substitutions K75L + H76L of the polypeptide of SEQ ID NO: 29.
In another embodiment, the xylanase comprises or consists of the substitutions K75L + I155M of the polypeptide of SEQ ID NO: 29.
In another embodiment, the xylanase comprises or consists of the substitutions K75L + V208L of the polypeptide of SEQ ID NO: 29.
In another embodiment, the xylanase comprises or consists of the substitutions H76L + I155M of the polypeptide of SEQ ID NO: 29. In another embodiment, the xylanase comprises or consists of the substitutions H76L + V208L of the polypeptide of SEQ ID NO: 29.
In another embodiment, the xylanase comprises or consists of the substitutions I155M + V208L of the polypeptide of SEQ ID NO: 29.
The xylanase may further comprise one or more additional alterations at one or more ( e.g ., several) other positions. The xylanase may further comprise one or more amino acid substitutions. The 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 1-50 amino acids such as 1-40 amino acids or 1-30 amino acides; 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.
Examples of 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. Common substitutions are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu/Val, Asn/Gln, Gln/Glu, Ala/Glu, and Asp/Gly. Other examples of conservative substitutions are G to A; A to G, S; V to I, L, A, T, S; I to V, L, M; L to I, M, V; M to L, I, V; P to A, S, N; F to Y, W, H; Y to F, W, H; W to Y, F, H; R to K, E, D; K to R, E, D; H to Q, N, S; D to N, E, K, R, Q; E to Q, D, K, R, N; S to T, A; T to S, V, A; C to S, T, A; N to D, Q, H, S; Q to E, N, H, K, R.
Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.
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 mutant molecules are tested for xylanase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et ai., 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, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et at, 1992, Science 255: 306-312; Smith et a!., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et at, 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.
In embodiment, the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 23; q) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 24; r) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 25; s) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 26; t) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 27; u) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 28; v) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 29; w) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 30; x) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 31 ; y) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 32; z) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 33; aa) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 34; bb) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 35; cc) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 36; dd) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 37; ee) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 38; ft) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 39; and gg) a polypeptide having 0-50, such as 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 40.
In an embodiment, the xylanase is obtained or obtainable from the taxonomic order Bacillales, preferably the taxonimic family Bacillaceae, or more preferably from the genus Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis or Paenibacillus pabuli. In one embodiment, the xylanase is obtained or obtainable from the taxonomic order Bacillales, preferably the taxonimic family. In an embodiment, theparent xylanase is a GH30 subfamily 8 xylanase.
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. Examples of 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. Biotechnol. 76: 245-251 ; Rasmussen- Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.
The xylanase may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the xylanse 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 xylanase is secreted extracellularly. The polypeptide may be a bacterial polypeptide. For example, the polypeptide may be a Gram-positive bacterial polypeptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces polypeptide having xylanase activity. In one embodiment, the polypeptide is from a bacterium of the class Bacilli, such as from the order Bacillales, or from the family Paenibacillaceae, or from the genus Paenibacillus or from the species Paenibacillus sp-19179 or Paenibacillus panacisoli.
In another embodiment, the polypeptide is from a bacterium of the class Clostridia, such as from the order Clostridiales, or from the family Clostridiaceae, or from the genus Clostridium or from the species Clostridium saccharobutylicum.
In another embodiment, the polypeptide is from a bacterium of the class Clostridia, such as from the order Clostridiales, or from the family Ruminococcaceae, or from the genus Ruminococcus, or from the species Ruminococcus sp. CAG:330.
In another embodiment, the polypeptide is from a bacterium of the class
Gammaproteobacteria, such as from the order Alteromonadales, or from the family Pseudoalteromonadaceae, or from the genus Pseudoalteromonas or from the species Pseudoalteromonas tetraodonis.
In another embodiment, the polypeptide is from a bacterium of the class
Gammaproteobacteria, such as from the order Enterobacteriales, or from the family Enterobacteriaceae, or from the genus Pectobacterium or from the species Pectobacterium carotovorum.
In another embodiment, the polypeptide is from a bacterium of the class Actinobacteria, such as from the order Streptomycetales, or from the family Streptomycetaceae, or from the genus Streptomyces or from the species Streptomyces sp-62627.
In another embodiment, the polypeptide is from a bacterium of the class
Gammaproteobacteria, such as from the order Vibrionales, or from the family Vibrionaceae, or from the genus Vibrio or from the species Vibrio rhizosphaerae.
In one aspect, the xylanse is a Bacillus al alophilus, 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, or Bacillus thuringiensis xylanase.
In another aspect, the xylanase is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus xylanase.
It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents. Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
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.
Alpha-galactosidases of the Invention
A polypeptide having alpha-galactosidase activity of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide 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 polypeptide obtained from a given source is secreted extracellularly.
In one embodiment, the polypeptide is from a bacterium of the class Bacilli, such as from the order Bacillales, or from the family Bacillaceae, or from the genus Bacillus or from the species Bacillus acidopullulyticus, Bacillus sp-19140 or Bacillus deramificans. In another embodiment, the polypeptide is from a bacterium of the class Bacilli, such as from the order Bacillales, or from the family Bacillaceae, or from the genus Anoxybacillus or from the species Anoxybacillus bogrovensis.
In a further embodiment, the polypeptide is from a fungus of the class Eurotiomycetes, such as from the order Eurotiales, or from the family Aspergillaceae, or from the genus Aspergillus or from the species Aspergillus unguis or Aspergillus puniceus.
It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL). 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. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), 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 invention relates to a use of a combination of a polypeptide having alpha- galactosidase activity and a polypeptide having xylanase activity in an animal feed or animal feed additive, wherein the polypeptide having alpha-galactosidase activity is a GH36 alpha- galactosidase and wherein the polypeptide having xylanase activity is a GH30 xylanase.
The polypeptide having alpha-galactosidase activity is a GH36 alpha-galactosidase typically selected from the group consisting of a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide having at least 90% sequence identity to SEQ ID NO: 9; g) a polypeptide having at least 90% sequence identity to SEQ ID NO: 11 ; h) a polypeptide having at least 90% sequence identity to SEQ ID NO: 12; i) a polypeptide having at least 90% sequence identity to SEQ ID NO: 14; j) a polypeptide having at least 90% sequence identity to SEQ ID NO: 15; k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16;
L) a polypeptide having at least 90% sequence identity to SEQ ID NO: 18; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; n) a polypeptide having at least 90% sequence identity to SEQ ID NO: 21 ; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO: 22
More typically, the polypeptide having alpha-galactosidase activity is a GH36 alpha- galactosidase selected from the group consisting of a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide having at least 90% sequence identity to SEQ ID NO: 9; k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; n) a polypeptide having at least 90% sequence identity to SEQ ID NO: 21 ; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO: 22
In a preferred embodiment of the invention, the polypeptide having alpha-galactosidase activity is a polypeptide selected from the group consisting of k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO: 22
The polypeptide having alpha-galactosidase activity is a GH36 alpha-galactosidase typically selected from the group consisting of a) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 2; b) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 3; c) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 5; d) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 6; e) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 8; f) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 9; g) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30amino acid substitutions, deletions or additions, compared to SEQ ID NO: 11 ; h) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 12; i) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 14; j) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 15; k) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 16;
L) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 18; m) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 19; n) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 21 ; and o) a polypeptide having 0-80, such as 0-70, 0-60, 0-50, 0-40 or 0-30 amino acid substitutions, deletions or additions, compared to SEQ ID NO: 22.
Enzyme Compositions
In an embodiment, the composition comprises the polypeptides of the invention and one or more formulating agents, as described below.
The compositions 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-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectinesterase, triacylglycerol lipase, xylanase, beta-xylosidase or any combination thereof.
The compositions may further comprise one or more microbes. In an embodiment, the microbe is selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium sp., Carnobacterium sp., Clostridium butyricum, Clostridium sp., Enterococcus faecium, Enterococcus sp., Lactobacillus sp., Lactobacillus acidophilus, Lactobacillus farciminus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, Lactococcus lactis, Lactococcus sp., Leuconostoc sp., Megasphaera elsdenii, Megasphaera sp., Pediococsus acidilactici, Pediococcus sp., Propionibacterium thoenii, Propionibacterium sp. and Streptococcus sp. or any combination thereof.
In an embodiment, the 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.
In an embodiment, the composition comprises one or more components selected from the list consisting of vitamins, minerals and amino acids. In an embodiment, the composition comprises plant based material from the sub-family Panicoideae as disclosed herein, preferably maize, corn, sorghum, switchgrass, millet, pearl millet, foxtail millet or in a processed form such as milled corn, milled maize, defatted maize, defatted destarched maize, milled sorghum, milled switchgrass, milled millet, milled foxtail millet, milled pearl millet, or any combination thereof.
The enzyme combination of the invention may be formulated as a liquid or a solid. For a liquid formulation, the formulating agent may comprise a polyol (such as e.g. glycerol, ethylene glycol or propylene glycol), a salt (such as e.g. sodium chloride, sodium benzoate, potassium sorbate) or a sugar or sugar derivative (such as e.g. dextrin, glucose, sucrose, and sorbitol). Thus in one embodiment, the composition is a liquid composition comprising the polypeptide of the invention and one or more formulating agents selected from the list consisting of glycerol, ethylene glycol, 1 ,2-propylene glycol, 1 ,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, dextrin, glucose, sucrose, and sorbitol. The liquid formulation may be sprayed onto the feed after it has been pelleted or may be added to drinking water given to the animals.
For a solid formulation, the formulation may be for example as a granule, spray dried powder or agglomerate (e.g. as disclosed in W02000/70034). The formulating agent may comprise a salt (organic or inorganic zinc, sodium, potassium or calcium salts such as e.g. such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol).
In one embodiment, the composition is a solid composition, such as a spray dried composition, comprising the xylanase of the invention and one or more formulating agents selected from the list consisting of sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch and cellulose. In a preferred embodiment, the formulating agent is selected from one or more of the following compounds: sodium sulfate, dextrin, cellulose, sodium thiosulfate, magnesium sulfate and calcium carbonate.
The present invention also relates to enzyme granules/particles comprising the combination of the invention optionally combined with one or more additional enzymes. The granule is composed of a core, and optionally one or more coatings (outer layers) surrounding the core.
Typically the granule/particle size, measured as equivalent spherical diameter (volume based average particle size), of the granule is 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm. The core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.
Methods for preparing the core can be found in Handbook of Powder Technology; Particle size enlargement by C. E. Capes; Volume 1; 1980; Elsevier. Preparation methods include known feed and granule formulation technologies, e.g. : a) spray dried products, wherein a liquid enzyme-containing solution is atomized in a spray drying tower to form small droplets which during their way down the drying tower dry to form an enzyme-containing particulate material; b) layered products, wherein the enzyme is coated as a layer around a pre-formed inert core particle, wherein an enzyme-containing solution is atomized, typically in a fluid bed apparatus wherein the pre-formed core particles are fluidized, and the enzyme-containing solution adheres to the core particles and dries up to leave a layer of dry enzyme on the surface of the core particle. Particles of a desired size can be obtained this way if a useful core particle of the desired size can be found. This type of product is described in, e.g., WO 97/23606; c) absorbed core particles, wherein rather than coating the enzyme as a layer around the core, the enzyme is absorbed onto and/or into the surface of the core. Such a process is described in WO 97/39116; d) extrusion or pelletized products, wherein an enzyme-containing paste is pressed to pellets or under pressure is extruded through a small opening and cut into particles which are subsequently dried. Such particles usually have a considerable size because of the material in which the extrusion opening is made (usually a plate with bore holes) sets a limit on the allowable pressure drop over the extrusion opening. Also, very high extrusion pressures when using a small opening increase heat generation in the enzyme paste, which is harmful to the enzyme; e) prilled products, wherein an enzyme-containing powder is suspended in molten wax and the suspension is sprayed, e.g., through a rotating disk atomiser, into a cooling chamber where the droplets quickly solidify (Michael S. Showell (editor); Powdered detergents·, Surfactant Science Series; 1998; vol. 71 ; page 140-142; Marcel Dekker). The product obtained is one wherein the enzyme is uniformly distributed throughout an inert material instead of being concentrated on its surface. Also US 4,016,040 and US 4,713,245 are documents relating to this technique; f) mixer granulation products, wherein a liquid is added to a dry powder composition of, e.g., conventional granulating components, the enzyme being introduced either via the liquid or the powder or both. The liquid and the powder are mixed and as the moisture of the liquid is absorbed in the dry powder, the components of the dry powder will start to adhere and agglomerate and particles will build up, forming granulates comprising the enzyme. Such a process is described in US 4,106,991 and related documents EP 170360, EP 304332, EP 304331 , WO 90/09440 and WO 90/09428. In a particular product of this process wherein various high-shear mixers can be used as granulators, granulates consisting of enzyme as enzyme, fillers and binders etc. are mixed with cellulose fibres to reinforce the particles to give the so-called T- granulate. Reinforced particles, being more robust, release less enzymatic dust; g) size reduction, wherein the cores are produced by milling or crushing of larger particles, pellets, tablets, briquettes etc. containing the enzyme. The wanted core particle fraction is obtained by sieving the milled or crushed product. Over and undersized particles can be recycled. Size reduction is described in (Martin Rhodes (editor); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons); h) fluid bed granulation, which involves suspending particulates in an air stream and spraying a liquid onto the fluidized particles via nozzles. Particles hit by spray droplets get wetted and become tacky. The tacky particles collide with other particles and adhere to them and form a granule; i) the cores may be subjected to drying, such as in a fluid bed drier. Other known methods for drying granules in the feed or detergent industry can be used by the skilled person. The drying preferably takes place at a product temperature of from 25 to 90°C. For some enzymes it is important the cores comprising the enzyme contain a low amount of water before coating. If water sensitive enzymes are coated before excessive water is removed, it will be trapped within the core and it may affect the activity of the enzyme negatively. After drying, the cores preferably contain 0.1-10 % w/w water.
The core may include additional materials such as fillers, fibre materials (cellulose or synthetic fibres), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.
The core may include a binder, such as synthetic polymer, wax, fat, or carbohydrate.
The core may include a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and/or an acidic buffer component, typically as a homogenous blend.
In one embodiment, the core comprises a material selected from the group consisting of salts (such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol), sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol), small organic molecules, starch, flour, cellulose and minerals and clay minerals (also known as hydrous aluminium phyllosilicates). In one embodiment, the core comprises a clay mineral such as kaolinite or kaolin.
The core may include an inert particle with the enzyme absorbed into it, or applied onto the surface, e.g., by fluid bed coating.
The core may have a diameter of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.
The core may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule. The optional coating(s) may include a salt and/or wax and/or flour coating, or other suitable coating materials.
The coating may be applied in an amount of at least 0.1% by weight of the core, e.g., at least 0.5%, 1% or 5%. The amount may be at most 100%, 70%, 50%, 40% or 30%.
The coating is preferably at least 0.1 pm thick, particularly at least 0.5 pm, at least 1 pm or at least 5 pm. In some embodiments the thickness of the coating is below 100 pm, such as below 60 pm, or below 40 pm.
The coating should encapsulate the core unit by forming a substantially continuous layer. A substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit is encapsulated or enclosed with few or no uncoated areas. The layer or coating should in particular be homogeneous in thickness.
The coating can further contain other materials as known in the art, e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.
A salt coating may comprise at least 60% by weight of a salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.
The salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles are less than 50 pm, such as less than 10 pm or less than 5 pm.
The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular having a solubility at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g per 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 g per 100 g water.
The salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminium. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, sorbate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used.
The salt in the coating may have a constant humidity at 20°C above 60%, particularly above 70%, above 80% or above 85%, or it may be another hydrate form of such a salt (e.g., anhydrate). The salt coating may be as described in W01997/05245, W01998/54980, W01998/55599, W02000/70034, W02006/034710, W02008/017661 , W02008/017659,
W02000/020569, W02001/004279, W01997/05245, W02000/01793, W02003/059086,
W02003/059087, W02007/031483, W02007/031485, W02007/044968, WO2013/192043, WO2014/014647 and WO2015/197719 or polymer coating such as described in WO 2001/00042.
Specific examples of suitable salts are NaCI (CH20°C=76%), Na2C03 (CH20°C=92%), NaN03 (CH20°C=73%), Na2HP04 (CH20°C=95%), Na3P04 (CH25°C=92%), NH4CI (CH20°C = 79.5%), (NH4)2HP04 (CH20°C = 93,0%), NH4H2P04 (CH20°C = 93.1%), (NH4)2S04 (CH20°C=81.1%), KCI (CH20°C=85%), K2HP04 (CH20°C=92%), KH2P04 (CH20°C=96.5%), KN03 (CH20°C=93.5%), Na2S04 (CH20°C=93%), K2S04 (CH20°C=98%), KHS04
(CH20°C=86%), MgS04 (CH20°C=90%), ZnS04 (CH20°C=90%) and sodium citrate (CH25°C=86%). Other examples include NaH2P04, (NH4)H2P04, CuS04, Mg(N03)2, magnesium acetate, calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, sodium acetate, sodium benzoate, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate and zinc sorbate.
The salt may be in anhydrous form, or it may be a hydrated salt, i.e. a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99/32595. Specific examples include anhydrous sodium sulfate (Na2S04), anhydrous magnesium sulfate (MgS04), magnesium sulfate heptahydrate (MgS04.7H20), zinc sulfate heptahydrate (ZnS04.7H20), sodium phosphate dibasic heptahydrate (Na2HP04.7H20), magnesium nitrate hexahydrate (Mg(N03)2(6H20)), sodium citrate dihydrate and magnesium acetate tetrahydrate.
Preferably the salt is applied as a solution of the salt, e.g., using a fluid bed.
A wax coating may comprise at least 60% by weight of a wax, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.
Specific examples of waxes are polyethylene glycols; polypropylenes; Carnauba wax; Candelilla wax; bees wax; hydrogenated plant oil or animal tallow such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC), polyvinyl alcohol (PVA), hydrogenated ox tallow, hydrogenated palm oil, hydrogenated cotton seeds and/or hydrogenated soy bean oil; fatty acid alcohols; mono-glycerides and/or di-glycerides, such as glyceryl stearate, wherein stearate is a mixture of stearic and palmitic acid; micro-crystalline wax; paraffin’s; and fatty acids, such as hydrogenated linear long chained fatty acids and derivatives thereof. A preferred wax is palm oil or hydrogenated palm oil.
The granule may comprise a core comprising the xylanase of the invention, one or more salt coatings and one or more wax coatings. Examples of enzyme granules with multiple coatings are shown in WO1993/07263, WO1997/23606 and WO2016/149636.
Non-dusting granulates may be produced, e.g., as disclosed in U.S. Patent Nos. 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art. The coating materials can be waxy coating materials and film-forming coating materials. Examples of waxy coating materials are polyethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591.
The granulate may further comprise one or more additional enzymes. Each enzyme will then be present in more granules securing a more uniform distribution of the enzymes, and also reduces the physical segregation of different enzymes due to different particle sizes. Methods for producing multi-enzyme co-granulates is disclosed in the ip.com disclosure IPCOM000200739D.
Another example of formulation of enzymes by the use of co-granulates is disclosed in WO 2013/188331.
The present invention also relates to protected enzymes prepared according to the method disclosed in EP 238,216.
Thus, in a further aspect, the present invention provides a granule, which comprises:
(a) a core comprising an xylanase according to the invention, and
(b) a coating consisting of one or more layer(s) surrounding the core.
In one embodiment, the coating comprises a salt coating as described herein. In one embodiment, the coating comprises a wax coating as described herein. In one embodiment, the coating comprises a salt coating followed by a wax coating as described herein.
Animal Feed Additives
An animal feed additive comprising a polypeptide having alpha-galactosidase activity and a polypeptide having xylanase activity; wherein the polypeptide having alpha-galactosidase activity is a GH36 alpha-galactosidase and wherein the polypeptide having xylanase activity is a GH30 xylanase. In a typical embdodument, the animal feed additive comprises a polypeptide having alpha-galactosidase activity wherein the GH36 alpha-galactosidase selected from the group consisting of a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide having at least 90% sequence identity to SEQ ID NO: 9; g) a polypeptide having at least 90% sequence identity to SEQ ID NO: 11 ; h) a polypeptide having at least 90% sequence identity to SEQ ID NO: 12; i) a polypeptide having at least 90% sequence identity to SEQ ID NO: 14; j) a polypeptide having at least 90% sequence identity to SEQ ID NO: 15; k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16;
L) a polypeptide having at least 90% sequence identity to SEQ ID NO: 18; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; n) a polypeptide having at least 90% sequence identity to SEQ ID NO: 21 ; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO: 22 and wherein the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; q) a polypeptide having at least 90% sequence identity to SEQ ID NO: 24; r) a polypeptide having at least 90% sequence identity to SEQ ID NO: 25; s) a polypeptide having at least 90% sequence identity to SEQ ID NO: 26; t) a polypeptide having at least 90% sequence identity to SEQ ID NO: 27; u) a polypeptide having at least 90% sequence identity to SEQ ID NO: 28; v) a polypeptide having at least 90% sequence identity to SEQ ID NO: 29; w) a polypeptide having at least 90% sequence identity to SEQ ID NO: 30; x) a polypeptide having at least 90% sequence identity to SEQ ID NO: 31 ; y) a polypeptide having at least 90% sequence identity to SEQ ID NO: 32; z) a polypeptide having at least 90% sequence identity to SEQ ID NO: 33; aa) a polypeptide having at least 90% sequence identity to SEQ ID NO: 34; bb) a polypeptide having at least 90% sequence identity to SEQ ID NO: 35; cc) a polypeptide having at least 90% sequence identity to SEQ ID NO: 36; dd) a polypeptide having at least 90% sequence identity to SEQ ID NO: 37; ee) a polypeptide having at least 90% sequence identity to SEQ ID NO: 38; ff) a polypeptide having at least 90% sequence identity to SEQ ID NO: 39; and gg) a polypeptide having at least 90% sequence identity to SEQ ID NO: 40.
Suitably, the animal feed additive comprises a polypeptide having alpha-galactosidase activity wherein the GH36 alpha-galactosidase selected from the group consisting of a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide having at least 90% sequence identity to SEQ ID NO: 9; k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; n) a polypeptide having at least 90% sequence identity to SEQ ID NO: 21 ; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO: 22 and wherein the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; и) a polypeptide having at least 90% sequence identity to SEQ ID NO: 29; and gg) a polypeptide having at least 90% sequence identity to SEQ ID NO: 40.
In a further suitable embodiment, the animal feed additive comprises a polypeptide having alpha-galactosidase activity wherein the GH36 alpha-galactosidase selected from the group consisting of к) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO: 22 and wherein the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; u) a polypeptide having at least 90% sequence identity to SEQ ID NO: 29; and gg) a polypeptide having at least 90% sequence identity to SEQ ID NO: 40.
In a preferred embodiment, the animal feed additive comprises wherein a polypeptide having alpha-galactosidase activity selected from k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; and a polypeptide having xylanase activity selected from p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23.
The animal feed additive may further comprise one or more additional components selected from the group consisting of: one or more vitamins; one or more minerals; one or more amino acids; one or more phytogenies; one or more prebiotics; one or more organic acids; and one or more other feed ingredients.
The animal feed additive typically comprises an amount of alpha-galactosidase and xylanase so as to increase the Average Metabolizable Energy of plant-based diet. An interesting aspect of the invention is directed to a method of improving the Average Metabolizable Energy of plant-based diet in a monogastric animal comprising administering an animal feed additive, composition, or combination as defined herein.
In an embodiment, the animal feed or animal feed additive comprises a formulating agent and the enzyme combination of the invention. In a further embodiment, the formulating agent comprises one or more of the following compounds: 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 and cellulose.
Thus, the invention further relates to an animal feed additive optionally comprising one or more vitamins and the enzyme combination of the invention. The invention also relates to an animal feed additive comprising one or more minerals and the enzyme combination of the invention. The invention also relates to an animal feed additive comprising one or more amino acids and a xylanase variant of the invention.
In an embodiment, the amount of each of the enzymes in the animal feed additive is between 0.001% and 10% by weight of the composition.
In an embodiment, the animal feed additive comprises one or more formulating agents, preferably as described herein above.
In an embodiment, the animal feed additive comprises one or more additional enzymes, preferably as described herein below.
In an embodiment, the animal feed additive comprises one or more probiotics, preferably as described herein below.
In an embodiment, the animal feed additive comprises one or more vitamins, preferably as described herein below.
In an embodiment, the animal feed additive comprises one or more minerals, preferably as described herein below.
In an embodiment, the animal feed additive comprises one or more amino acids, preferably as described herein below.
In an embodiment, the animal feed additive comprises one or more prebiotics, preferably as described herein below. In an embodiment, the animal feed additive comprises one or more organic acids, preferably as described herein below.
In an embodiment, the animal feed additive comprises one or more phytogenies, preferably as described herein below.
Animal Feed
The present invention also relates to animal feed compositions comprising the combination or animal feed of the invention. The invention also relates to an animal feed comprising the granule as described herein and plant based material. The invention also relates to an animal feed comprising the animal feed additive as described herein and plant based material. In one embodiment, the plant based material is from the sub-family Panicoideae.
Animal feed compositions or diets have a relatively high content of protein. Poultry and pig diets can be characterised as indicated in Table B of WO 01/58275, columns 2-3. Fish diets can be characterised as indicated in column 4 of this Table B. Furthermore such fish diets usually have a crude fat content of 200-310 g/kg.
An animal feed composition according to the invention has a crude protein content of 50- 800 g/kg, and furthermore comprises at least one xylanase as claimed herein.
Furthermore, or in the alternative (to the crude protein content indicated above), the animal feed composition of the invention has a content of metabolisable energy of 10-30 MJ/kg; and/or a content of calcium of 0.1-200 g/kg; and/or a content of available phosphorus of 0.1-200 g/kg; and/ora content of methionine of 0.1-100 g/kg; and/or a content of methionine plus cysteine of 0.1-150 g/kg; and/or a content of lysine of 0.5-50 g/kg.
In particular embodiments, the content of metabolisable energy, crude protein, calcium, phosphorus, methionine, methionine plus cysteine, and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO 01/58275 (R. 2-5).
Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25, i.e. Crude protein (g/kg)= N (g/kg) x 6.25. The nitrogen content is determined by the Kjeldahl method (A.O.A.C., 1984, Official Methods of Analysis 14th ed., Association of Official Analytical Chemists, Washington DC).
Metabolisable energy can be calculated on the basis of the NRC publication Nutrient requirements in swine, ninth revised edition 1988, subcommittee on swine nutrition, committee on animal nutrition, board of agriculture, national research council. National Academy Press, Washington, D.C., pp. 2-6, and the European Table of Energy Values for Poultry Feed-stuffs, Spelderholt centre for poultry research and extension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen & looijen bv, Wageningen. ISBN 90-71463-12-5.
The dietary content of calcium, available phosphorus and amino acids in complete animal diets is calculated on the basis of feed tables such as Veevoedertabel 1997, gegevens over chemische samenstelling, verteerbaarheid en voederwaarde van voedermiddelen, Central Veevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.
In a particular embodiment, the animal feed composition of the invention contains at least one vegetable protein as defined above.
The animal feed composition of the invention may also contain animal protein, such as Meat and Bone Meal, Feather meal, and/or Fish Meal, typically in an amount of 0-25%. The animal feed composition of the invention may also comprise Dried Distillers Grains with Solubles (DDGS), typically in amounts of 0-30%.
In still further particular embodiments, the animal feed composition of the invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or 0-20% whey.
The animal feed may comprise vegetable proteins. In particular embodiments, the protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (w/w). Vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example, materials from plants of the families Fabaceae ( Leguminosae ), Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupin meal, rapeseed meal, and combinations thereof.
In a particular embodiment, the vegetable protein source is material from one or more plants of the family Fabaceae, e.g., soybean, lupine, pea, or bean. In another particular embodiment, the vegetable protein source is material from one or more plants of the family Chenopodiaceae, e.g. beet, sugar beet, spinach or quinoa. Other examples of vegetable protein sources are rapeseed, and cabbage. In another particular embodiment, soybean is a preferred vegetable protein source. Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, and sorghum.
Animal diets can e.g. be manufactured as mash feed (non-pelleted) or pelleted feed. Typically, the milled feed-stuffs are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species in question. Enzymes can be added as solid or liquid enzyme formulations. For example, for mash feed a solid or liquid enzyme formulation may be added before or during the ingredient mixing step. For pelleted feed the (liquid or solid) xylanase/enzyme preparation may also be added before or during the feed ingredient step. Typically a liquid xylanase/enzyme preparation comprises the xylanase of the invention optionally with a polyol, such as glycerol, ethylene glycol or propylene glycol, and is added after the pelleting step, such as by spraying the liquid formulation onto the pellets. The enzyme may also be incorporated in a feed additive or premix.
Alternatively, the xylanase can be prepared by freezing a mixture of liquid enzyme solution with a bulking agent such as ground soybean meal, and then lyophilizing the mixture. The final enzyme concentration in the diet is within the range of 0.01-200 mg enzyme protein per kg diet, preferably between 0.05-100 mg/kg diet, more preferably 0.1-50 mg, even more preferably 0.2-20 mg enzyme protein per kg animal diet.
It is at present contemplated that the enzyme is administered in one or more of the following amounts (dosage ranges): 0.01-200; 0.05-100; 0.1-50; 0.2-20; 0.1-1 ; 0.2-2; 0.5-5; or 1- 10; - all these ranges being in mg xylanase protein per kg feed (ppm).
For determining mg xylanase protein per kg feed, the xylanase is purified from the feed composition, and the specific activity of the purified xylanase is determined using a relevant assay (see under xylanase activity). The xylanase activity of the feed composition as such is also determined using the same assay, and on the basis of these two determinations, the dosage in mg xylanase protein per kg feed is calculated.
In a particular embodiment, the animal feed additive of the invention is intended for being included (orprescribed as having to be included) in animal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05 to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed). This is so in particular for premixes.
The same principles apply for determining mg xylanase protein in feed additives. Of course, if a sample is available of the xylanase used for preparing the feed additive or the feed, the specific activity is determined from this sample (no need to purify the xylanase from the feed composition or the additive).
Use in Animal Feed
The present invention is also directed to methods for using the animal feed additive in animal feed.
The term animal includes all animals. Examples of animals are non-ruminants, and ruminants. Ruminant animals include, for example, animals such as sheep, goats, and cattle, e.g., beef cattle, cows, and young calves. In a particular embodiment, the animal is a nonruminant animal. 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; and fish (including but not limited to salmon, trout, tilapia, catfish and carps; and crustaceans (including but not limited to shrimps and prawns).
In the use according to the invention the animal feed additive can be fed to the animal before, after, or simultaneously with the diet. The latter is preferred. A well-defined additive preparation is advantageous. For instance, it is much easier to dose correctly to the feed a xylanase that is essentially free from interfering or contaminating other xylanases. The term dose correctly refers in particular to the objective of obtaining consistent and constant results, and the capability of optimizing dosage based upon the desired effect.
The animal feed additive can be (a) added directly to the feed, or (b) it can be used in the production of one or more intermediate compositions such as premixes that are subsequently added to the feed (or used in a treatment process).
Methods for Improving the Nutritional Value of Animal Feed
The present invention further relates to a method for improving the nutritional value of an animal feed comprising plant based material from the sub-family Panicoideae, comprising adding to the feed an animal feed additive.
The term improving the nutritional value of an animal feed means improving the availability of nutrients in the feed. The nutritional values refer in particular to improving the solubilization and degradation of the arabinoxylan-containing fraction (e.g., such as hemicellulose) of the feed, thereby leading to increased release of nutrients from cells in the endosperm that have cell walls composed of highly recalcitrant hemicellulose. Consequently, an increased release of arabinoxylan oligomers indicates a disruption of the cell walls and as a result the nutritional value of the feed is improved resulting in increased growth rate and/or weight gain and/or feed conversion (i.e., the weight of ingested feed relative to weight gain). In addition the arabinoxylan oligomer release may result in improved utilization of these components per se either directly or by bacterial fermentation in the hind gut thereby resulting in a production of short chain fatty acids that may be readily absorbed in the hind and utilised in the energy metabolism.
Methods of Improving Animal Performance
The invention further relates to a method of improving one or more performance parameters of an animal, comprising administering to one or more animals a animal feed additive and plant based material from the sub-family Panicoideae.
The plant based material from the sub-family Panicoideae may be administered together or separately with the animal feed additve. In an embodiment, the plant based material from the sub-family Panicoideae is maize, corn, sorghum, switchgrass, millet, pearl millet, foxtail millet or in a processed form such as milled corn, milled maize, defatted maize, defatted destarched maize, milled sorghum, milled switchgrass, milled millet, milled foxtail millet, milled pearl millet, or any combination thereof. In a further embodiment, the the plant based material from the subfamily Panicoideae is from the seed fraction (such as endosperm and/or husk) of the plant.
In an embodiment, the improvement in the performance of an animal is an increase in body weight gain increases the Average Metabolizable Energy improved feed conversion ratio. In a further embodiment, the improvement is an increased feed efficiency. In a further embodiment, the improvement is an increase in body weight gain and/or an improved feed conversion ratio and/or an increased feed efficiency. In an embodiment, the improvement is the increase the Average Metabolizable Energy.
Methods of Solubilizing Xylan from Plant Based Materials
The invention further relates to methods of solubilizing xylan from a plant based material, e.g., by degrading the xylose backbone of sterically hindered arabinoxylan found in plant based material from the sub-family Panicoideae, thereby solubilizing increased amounts of arabinoxylan which is measured as arabinose and xylose. Increased degradation, and thereby increased arabinose and xylose release, can result in advantages for many industries which use plant based material from the sub-family Panicoideae.
The amount of starch present in untreated plant material makes it difficult to detect significant solubilization of arabinoxylan. Thus model substrates, wherein the starch and fat present in the plant material is removed without effecting the degree of substitution, can be used to aid the determination of improved enzyme combinations over known prior art combinations. One model substrate is defatted destarched maize (DFDSM) and can be prepared as described in the Examples. It is important that the model substrate is not prepared using strongly acidic or basic conditions or high temperatures, since such conditions can remove the side chain carbohydrate molecules and/or ester groups present on the xylan backbone. If these side chain groups are removed, then the complexity and degree of substitution will be reduced resulting in an arabinoxylan material which is easy to degrade by known solutions. It is for this reason that heat, acid and/or base pre-treatment is used in biomass conversion.
In order to measure the solubilization of the arabinoxylan, the soluble arabinoxylan is hydrolyzed with acid resulting in xylose and arabinose being released into the supernatant. This xylose and arabinose is then detected using, e.g., the HPLC method as described herein. The higher the degree of solubilization of the arabinoxylan, the higher the amount of xylose and arabinose released upon acid hydrolysis. It is believed that increasing the solubilization of the arabinoxylan opens up the cell walls that can result in the nutrients, such as starch and protein, which are trapped inside being released. The release of starch and other nutrients can result in improved animal performance and/or improve the nutritional value of an animal feed.
Methods of Releasing Starch
The invention further relates to a method of releasing starch from plant based material, comprising treating plant based material from the sub-family Panicoideae with the enzyme combination.
In one embodiment, the plant based material from the sub-family Panicoideae is from the tribe Andropogoneae such as the rank Andropogon or Andropterum or Apluda or Apocopis or Arthraxon or Bothriochloa or Capillipedium or Chionachne or Chrysopogon or Coelorachis or Coix or Cymbopogon or Dichanthium or Diheteropogon or Dimeria or Elionurus or Eremochloa or Euclasta or Eulalia or Germainia or Hemarthria or Heteropholis or Heteropogon or Hyparrhenia or Hyperthelia or Imperata or Ischaemum or Iseilema or Kerriochloa or Microstegium or Miscanthidium or Miscanthus or Mnesithea or Ophiuros or Oxyrhachis or Phacelurus or Pholiurus or Pogonatherum or Polytoca or Polytrias or Pseudopogonatherum or Pseudosorghum or Rhytachne or Rottboellia or Saccharum (e.g. sugar cane) or Sarga or Schizachyrium or Sehima or Sorghastrum or Sorghum or Spodiopogon or Thaumastochloa or Thelepogon or Themeda or Trachypogon or Triarrhena or Tripsacum or Urelytrum or Vetiveria or Vossia or Xerochloa or Zea.
In a preferred embodiment, the plant based material from the sub-family Panicoideae is from the rank Zea, such as the species Zea diploperennis, Zea luxurians, Zea mays, Zea nicaraguensis or Zea perennis.
In a preferred embodiment, the plant based material from the sub-family Panicoideae is from the rank Sorghum, such as the species Sorghum amplum, Sorghum angustum, Sorghum arundinaceum, Sorghum australiense, Sorghum bicolor, Sorghum brachypodum, Sorghum bulbosum, Sorghum ecarinatum, Sorghum exstans, Sorghum grande, Sorghum halepense, Sorghum hybrid cultivar, Sorghum interjectum, Sorghum intrans, Sorghum laxiflorum, Sorghum leiocladum, Sorghum macrospermum, Sorghum matarankense, Sorghum nitidum, Sorghum plumosum, Sorghum propinquum, Sorghum purpureosericeum, Sorghum stipoideum, Sorghum sudanense, Sorghum timorense, Sorghum versicolor, Sorghum sp. 'Silk' or Sorghum sp. as defined in W02007/002267.
In another embodiment, the plant based material from the sub-family Panicoideae is from the tribe Paniceae such as the rank Acritochaete, Acroceras, Alexfloydia, Alloteropsis, Amphicarpum, Ancistrachne, Anthephora, Brachiaria (e.g. signal grass), Calyptochloa, Cenchrus, Chaetium, Chaetopoa, Chamaeraphis, Chlorocalymma, Cleistochloa, Cyphochlaena, Cyrtococcum, Dichanthelium, Digitaria, Dissochondrus, Echinochloa, Entolasia, Eriochloa, Homopholis, Hygrochloa, Hylebates, Ixophorus, Lasiacis, Leucophrys, Louisiella, Megaloprotachne, Megathyrsus, Melinis, Microcalamus, Moorochloa, Neurachne, Odontelytrum, Oplismenus, Ottochloa, Panicum, Paractaenum, Paraneurachne, Paratheria, Parodiophyllochloa, Paspalidium, Pennisetum, Plagiosetum, Poecilostachys, Pseudechinolaena, Pseudochaetochloa, Pseudoraphis, Rupichloa, Sacciolepis, Scutachne, Setaria, Setariopsis, Snowdenia, Spinifex, Stenotaphrum, Stereochlaena, Thrasya, Thuarea, Thyridolepis, Tricholaena, unclassified Paniceae, Uranthoecium, Urochloa (e.g. signal grass), Walwhalleya, Whiteochloa, Yakirra, Yvesia, Zuloagaea or Zygochloa.
In a preferred embodiment, the plant based material from the sub-family Panicoideae is from the rank Panicum, such as the species Panicum adenophorum, Panicum aff. aquaticum JKT-2012, Panicum amarum, Panicum antidotale, Panicum aquaticum, Panicum arctum, Panicum arundinariae, Panicum atrosanguineum, Panicum auricomum, Panicum auritum, Panicum bartlettii, Panicum bergii, Panicum bisulcatum, Panicum boliviense, Panicum brazzavillense, Panicum brevifolium, Panicum caaguazuense, Panicum campestre, Panicum capillare, Panicum cayennense, Panicum cayoense, Panicum cervicatum, Panicum chloroleucum, Panicum claytonii, Panicum coloratum, Panicum cyanescens, Panicum decompositum, Panicum deustum, Panicum dichotomiflorum, Panicum dinklagei, Panicum distichophyllum, Panicum dregeanum, Panicum elephantipes, Panicum fauriei, Panicum flexile, Panicum fluviicola, Panicum gouinii, Panicum gracilicaule, Panicum granuliferum, Panicum guatemalense, Panicum hallii, Panicum heterostachyum, Panicum hirticaule, Panicum hirtum, Panicum hylaeicum, Panicum incumbens, Panicum infestum, Panicum italicum, Panicum laetum, Panicum laevinode, Panicum lanipes, Panicum larcomianum, Panicum longipedicellatum, Panicum machrisianum, Panicum malacotrichum, Panicum margaritiferum, Panicum micranthum, Panicum miliaceum, Panicum milioides, Panicum millegrana, Panicum mystasipum, Panicum natalense, Panicum nephelophilum, Panicum nervosum, Panicum notatum, Panicum olyroides, Panicum paludosum, Panicum pansum, Panicum pantrichum, Panicum parvifolium, Panicum parviglume, Panicum pedersenii, Panicum penicillatum, Panicum petersonii, Panicum phragmitoides, Panicum piauiense, Panicum pilosum, Panicum pleianthum, Panicum polycomum, Panicum polygonatum, Panicum pseudisachne, Panicum pygmaeum, Panicum pyrularium, Panicum queenslandicum, Panicum racemosum, Panicum repens, Panicum rhizogonum, Panicum rigidulum, Panicum rivale, Panicum rude, Panicum rudgei, Panicum schinzii, Panicum schwackeanum, Panicum sellowii, Panicum seminudum, Panicum stapfianum, Panicum stenodes, Panicum stramineum, Panicum subalbidum, Panicum subtiramulosum, Panicum sumatrense, Panicum tenellum, Panicum tenuifolium, Panicum trichanthum, Panicum trichidiachne, Panicum trichoides, Panicum tricholaenoides, Panicum tuerckheimii, Panicum turgidum, Panicum urvilleanum, Panicum validum, Panicum venezuelae, Panicum verrucosum, Panicum virgatum, Panicum wettsteinii, Panicum sp., Panicum sp. Christin 16-200, Panicum sp. ELS-2011, Panicum sp. EM389 or Panicum sp. Forest 761.
In a further embodiment, the plant based material from the sub-family Panicoideae is maize (Zea), corn (Zea), sorghum (Sorghum), switchgrass ( Panicum virgatum), millet ( Panicum miliaceum), pearl millet ( Cenchrus violaceus also called Pennisetum glaucum), foxtail millet (Setaria italica also called Panicum italicum) or in a processed form such as milled corn, milled maize, defatted maize, defatted destarched maize, milled sorghum, milled switchgrass, milled millet, milled foxtail millet, milled pearl millet, or any combination thereof.
In an embodiment, the plant based material from the sub-family Panicoideae is from the seed of the plant. In a preferred embodiment, the plant based material from the sub-family Panicoideae is from the seed of maize (Zea), corn (Zea), sorghum (Sorghum), switchgrass (Panicum virgatum), millet (Panicum miliaceum), pearl millet (Cenchrus violaceus also called Pennisetum glaucum), foxtail millet (Setaria italica also called Panicum italicum) or wherein the seed has been processed such as milled corn, milled maize, defatted maize, defatted destarched maize, milled sorghum, milled switchgrass, milled millet, milled foxtail millet, milled pearl millet, or any combination thereof.
Additional Enzymes
In another embodiment, the compositions described herein optionally include one or more additional enzymes. Enzymes can be classified on the basis of the handbook Enzyme Nomenclature from NC-IUBMB, 1992), see also the ENZYME site at the internet: http://www.expasv.ch/enzvme/. ENZYME is a repository of information relative to the nomenclature of enzymes. It is primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB), Academic Press, Inc., 1992, and it describes each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided (Bairoch A. The ENZYME database, 2000, Nucleic Acids Res 28:304-305). This IUB-MB Enzyme nomenclature is based on their substrate specificity and occasionally on their molecular mechanism; such a classification does not reflect the structural features of these enzymes.
Another classification of certain glycoside hydrolase enzymes, such as endoglucanase, galactanase, mannanase, dextranase, lysozyme and galactosidase is described in Henrissat et al, “The carbohydrate-active enzymes database (CAZy) in 2013”, Nucl. Acids Res. (1 January 2014) 42 (D1): D490-D495; see also www.cazy.org.
Thus the composition of the invention may also comprise at least one other enzyme selected from the group comprising of acetylxylan esterase (EC 3.1.1.23), acylglycerol lipase (EC
3.1.1.72), alpha-amylase (EC 3.2.1.1), beta-amylase (EC 3.2.1.2), arabinofuranosidase (EC 3.2.1.55), cellobiohydrolases (EC 3.2.1.91), cellulase (EC 3.2.1.4), feruloyl esterase (EC
3.1.1 .73), galactanase (EC 3.2.1.89), alpha-galactosidase (EC 3.2.1.22), beta-galactosidase (EC 3.2.1.23), glucan 1 ,4-a-glucosidase (glucoamylase) (EC 3.2.1.3), glucan 1,4-alpha- maltohydrolase (maltogenic alpha-amylase) (EC 3.2.1.133), beta-glucanase (EC 3.2.1.6), beta- glucosidase (EC 3.2.1.21), triacylglycerol lipase (EC 3.1.1.3), lysophospholipase (EC 3.1.1.5), lysozyme (EC 3.2.1.17), alpha-mannosidase (EC 3.2.1.24), beta-mannosidase (mannanase) (EC 3.2.1.25), phytase (EC 3.1.3.8, EC 3.1.3.26, EC 3.1.3.72), phospholipase A1 (EC 3.1.1.32), phospholipase A2 (EC 3.1.1.4), phospholipase D (EC 3.1.4.4), protease (EC 3.4), pullulanase (EC 3.2.1.41), pectinesterase (EC 3.1.1.11), xylanase (EC 3.2.1.8, EC 3.2.1.136), beta- xylosidase (EC 3.2.1.37), or any combination thereof.
In a particular embodiment the composition of the invention comprises a galactanase (EC 3.2.1.89) and a beta-galactosidase (EC 3.2.1.23).
In a particular embodiment the composition of the invention comprises an alpha- galactosidase (EC 3.2.1.22). In a particular embodiment, the composition of the invention comprises a phytase (EC 3.1.3.8 or 3.1.3.26). Examples of commercially available phytases include Bio-Feed™ Phytase (Novozymes), Ronozyme® P, Ronozyme® NP and Ronozyme® HiPhos (DSM Nutritional Products), Natuphos™ (BASF), Natuphos™ E (BASF), Finase® and Quantum® Blue (AB Enzymes), OptiPhos® (Huvepharma), AveMix® Phytase (Aveve Biochem), Phyzyme® XP (Verenium/DuPont) and Axtra® PHY (DuPont). Other preferred phytases include those described in e.g. WO 98/28408, WO 00/43503, and WO 03/066847.
In a particular embodiment, the composition of the invention comprises a xylanase (EC 3.2.1.8). Examples of commercially available xylanases include Ronozyme® WX (DSM Nutritional Products), Econase® XT and Barley (AB Vista), Xylathin® (Verenium), Hostazym® X (Huvepharma), Axtra® XB (Xylanase/beta-glucanase, DuPont) and Axtra® XAP (Xylanase/amylase/protease, DuPont), AveMix® XG 10 (xylanase/glucanase) and AveMix® 02 CS (xylanase/glucanase/pectinase, Aveve Biochem), and Naturgrain (BASF).
In a particular embodiment, the composition of the invention comprises a protease (EC 3.4). Examples of commercially available proteases include Ronozyme® ProAct (DSM Nutritional Products).
In a particular embodiment, the composition of the invention comprises an alpha-amylase (EC 3.2.1.1). Examples of commercially available alpha-amylases include Ronozyme® A and RONOZYME® RumiStar™ (DSM Nutritional Products).
In one embodiment, the composition of the invention comprises a multicomponent enzyme product, such as FRA® Octazyme (Framelco), Ronozyme® G2, Ronozyme® VP and Ronozyme® MultiGrain (DSM Nutritional Products), Rovabio® Excel or Rovabio® Advance (Adisseo).
Eubiotics
Eubiotics are compounds which are designed to give a healthy balance of the micro-flora in the gastrointestinal tract. Eubiotics cover a number of different feed additives, such as probiotics, prebiotics, phytogenies (essential oils) and organic acids which are described in more detail below.
Probiotics
In an embodiment, the animal feed composition further comprises one or more additional probiotic. In a particular embodiment, the animal feed composition further comprises a bacterium from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium and Megasphaera or any combination thereof.
In a preferred embodiment, animal feed composition further comprises a bacterium from one or more of the following strains: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Enterococcus faecium, Enterococcus spp, and Pediococcus spp, Lactobacillus spp, Bifidobacterium spp, Lactobacillus acidophilus, Pediococsus acidilactici, Lactococcus lactis, Bifidobacterium bifidum, Propionibacterium thoenii, Lactobacillus farciminus, lactobacillus rhamnosus, Clostridium butyricum, Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Lactobacillus salivarius ssp. salivarius, Megasphaera elsdenii, Propionibacteria sp.
In a more preferred embodiment, composition, animal feed additive or animal feed further comprises a bacterium from one or more of the following strains of Bacillus subtilis: 3A-P4 (PTA- 6506), 15A-P4 (PTA-6507), 22C-P1 (PTA-6508), 2084 (NRRL B-500130), LSSA01 (NRRL-B- 50104), BS27 (NRRL B-501 05), BS 18 (NRRL B-50633), BS 278 (NRRL B-50634), DSM 29870, DSM 29871 , DSM 32315, NRRL B-50136, NRRL B-50605, NRRL B-50606, NRRL B-50622 and PTA-7547.
In a more preferred embodiment, composition, animal feed additive or animal feed further comprises a bacterium from one or more of the following strains of Bacillus pumilus: NRRL B- 50016, ATCC 700385, NRRL B-50885 or NRRL B-50886.
In a more preferred embodiment, composition, animal feed additive or animal feed further comprises a bacterium from one or more of the following strains of Bacillus lichenformis: NRRL B 50015, NRRL B-50621 or NRRL B-50623.
In a more preferred embodiment, composition, animal feed additive or animal feed further comprises a bacterium from one or more of the following strains of Bacillus amyloliquefaciens: DSM 29869, DSM 29869, NRRL B 50607, PTA-7543, PTA-7549, NRRL B-50349, NRRL B- 50606, NRRL B-50013, NRRL B-50151, NRRL B-50141 , NRRL B-50147 or NRRL B-50888.
The bacterial count of each of the bacterial strains in the animal feed composition is between 1x104 and 1x1014 CFU/kg of dry matter, preferably between 1x106 and 1x1012 CFU/kg of dry matter, and more preferably between 1x107 and 1x1011 CFU/kg of dry matter. In a more preferred embodiment the bacterial count of each of the bacterial strains in the animal feed composition is between 1x10® and 1x1010 CFU/kg of dry matter.
The bacterial count of each of the bacterial strains in the animal feed composition is between 1x105 and 1x1015 CFU/animal/day, preferably between 1x107 and 1x1013 CFU/animal/day, and more preferably between 1x10® and 1x1012 CFU/animal/day. In a more preferred embodiment the bacterial count of each of the bacterial strains in the animal feed composition is between 1x109 and 1x1011 CFU/animal/day. In one embodiment, the amount of probiotics is 0.001% to 10% by weight of the composition.
In another embodiment, the one or more bacterial strains are present in the form of a stable spore. Examples of commercial products are Cylactin® (DSM Nutritional Products), Alterion (Adisseo), Enviva PRO (DuPont Animal Nutrition), Syncra® (mix enzyme + probiotic, DuPont Animal Nutrition), Ecobiol® and Fecinor® (Norel/Evonik) and GutCare® PY1 (Evonik).
Prebiotics
Prebiotics are substances that induce the growth or activity of microorganisms (e.g., bacteria and fungi) that contribute to the well-being of their host. Prebiotics are typically non- digestible fiber compounds that pass undigested through the upper part of the gastrointestinal tract and stimulate the growth or activity of advantageous bacteria that colonize the large bowel by acting as substrate for them. Normally, prebiotics increase the number or activity of bifidobacteria and lactic acid bacteria in the Gl tract.
Yeast derivatives (inactivated whole yeasts or yeast cell walls) can also be considered as prebiotics. They often comprise mannan-oligosaccharids, yeast beta-glucans or protein contents and are normally derived from the cell wall of the yeast, Saccharomyces cerevisiae.
In one embodiment, the amount of prebiotics is 0.001% to 10% by weight of the composition. Examples of yeast products are Yang® and Agrimos (Lallemand Animal Nutrition).
Phytogenies
Phytogenies are a group of natural growth promoters or non-antibiotic growth promoters used as feed additives, derived from herbs, spices or other plants. Phytogenies can be single substances prepared from essential oils/extracts, essential oils/extracts, single plants and mixture of plants (herbal products) or mixture of essential oils/extracts/plants (specialized products).
Examples of phytogenies are rosemary, sage, oregano, thyme, clove, and lemongrass. Examples of essential oils are thymol, eugenol, meta-cresol, vaniline, salicylate, resorcine, guajacol, gingerol, lavender oil, ionones, irone, eucalyptol, menthol, peppermint oil, alpha-pinene; limonene, anethol, linalool, methyl dihydrojasmonate, carvacrol, propionic acid/propionate, acetic acid/acetate, butyric acid/butyrate, rosemary oil, clove oil, geraniol, terpineol, citronellol, amyl and/or benzyl salicylate, cinnamaldehyde, plant polyphenol (tannin), turmeric and curcuma extract.
In one embodiment, the amount of phytogeneics is 0.001% to 10% by weight of the composition. Examples of commercial products are Crina® (DSM Nutritional Products); Cinergy™, Biacid™, ProHacidTM Classic and ProHacidTM Advance™ (all Promivi/Cargill) and Envivo EO (DuPont Animal Nutrition).
Organic Acids
Organic acids (C1-C7) are widely distributed in nature as normal constituents of plants or animal tissues. They are also formed through microbial fermentation of carbohydrates mainly in the large intestine. They are often used in swine and poultry production as a replacement of antibiotic growth promoters since they have a preventive effect on the intestinal problems like necrotic enteritis in chickens and Escherichia coli infection in young pigs. Organic acids can be sold as mono component or mixtures of typically 2 or 3 different organic acids. Examples of organic acids are propionic acid, formic acid, citric acid, lactic acid, sorbic acid, malic acid, acetic acid, fumaric acid, benzoic acid, butyric acid and tartaric acid or their salt (typically sodium or potassium salt such as potassium diformate or sodium butyrate).
In one embodiment, the amount of organic acid is 0.001% to 10% by weight of the composition. Examples of commercial products are VevoVitall® (DSM Nutritional Products), Amasil®, Luprisil®, Lupro-Grain®, Lupro-Cid®, Lupro-Mix® (BASF), n-Butyric Acid AF (OXEA) and Adimix Precision (Nutriad).
Premix
The incorporation of the composition of feed additives as exemplified herein above to animal feeds, for example poultry feeds, is in practice carried out using a concentrate or a premix. A premix designates a preferably uniform mixture of one or more microingredients with diluent and/or carrier. Premixes are used to facilitate uniform dispersion of micro-ingredients in a larger mix. A premix according to the invention can be added to feed ingredients or to the drinking water as solids (for example as water soluble powder) or liquids.
Amino Acids
The composition of the invention may further comprise one or more amino acids. Examples of amino acids which are used in animal feed are lysine, alanine, beta-alanine, threonine, methionine and tryptophan. In one embodiment, the amount of amino acid is 0.001% to 10% by weight of the composition.
Vitamins and Minerals
In another embodiment, the animal feed may include one or more vitamins, such as one or more fat-soluble vitamins and/or one or more water-soluble vitamins. In another embodiment, the animal feed may optionally include one or more minerals, such as one or more trace minerals and/or one or more macro minerals.
Usually fat- and water-soluble vitamins, as well as trace minerals form part of a so-called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed.
Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, e.g., vitamin K3. Non-limiting examples of water-soluble vitamins include vitamin C, vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g., Ca-D- panthothenate.
Non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, iodine, selenium and zinc.
Non-limiting examples of macro minerals include calcium, magnesium, phosphorus, potassium and sodium.
In one embodiment, the amount of vitamins is 0.001% to 10% by weight of the composition. In one embodiment, the amount of minerals is 0.001% to 10% by weight of the composition.
The nutritional requirements of these components (exemplified with poultry and piglets/pigs) are listed in Table A of WO 01/58275. Nutritional requirement means that these components should be provided in the diet in the concentrations indicated.
In the alternative, the animal feed additive of the invention comprises at least one of the individual components specified in Table A of WO 01/58275. At least one means either of, one or more of, one, or two, or three, or four and so forth up to all thirteen, or up to all fifteen individual components. More specifically, this at least one individual component is included in the additive of the invention in such an amount as to provide an in-feed-concentration within the range indicated in column four, or column five, or column six of Table A. In a still further embodiment, the animal feed additive of the invention comprises at least one of the below vitamins, preferably to provide an in-feed-concentration within the ranges specified in the below Table 1 (for piglet diets, and broiler diets, respectively).
Table 1 : Typical vitamin recommendations
Figure imgf000049_0001
Figure imgf000050_0001
Other feed ingredients
The composition of the invention may further comprise colouring agents, stabilisers, growth improving additives and aroma compounds/flavourings, polyunsaturated fatty acids (PUFAs); reactive oxygen generating species, antioxidants, anti-microbial peptides, anti-fungal polypeptides and mycotoxin management compounds.
Examples of colouring agents are carotenoids such as beta-carotene, astaxanthin, and lutein.
Examples of aroma compounds/flavourings are creosol, anethol, deca-, undeca-and/or dodeca-lactones, ionones, irone, gingerol, piperidine, propylidene phatalide, butylidene phatalide, capsaicin and tannin.
Examples of antimicrobial peptides (AMP’s) are CAP18, Leucocin A, Tritrpticin, Protegrin- 1 , Thanatin, Defensin, Lactoferrin, Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000), Plectasins, and Statins, including the compounds and polypeptides disclosed in WO 03/044049 and WO 03/048148, as well as variants or fragments of the above that retain antimicrobial activity.
Examples of antifungal polypeptides (AFP’s) are the Aspergillus giganteus, and Aspergillus niger peptides, as well as variants and fragments thereof which retain antifungal activity, as disclosed in WO 94/01459 and WO 02/090384.
Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma- linoleic acid.
Examples of reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a syntethase.
Antioxidants can be used to limit the number of reactive oxygen species which can be generated such that the level of reactive oxygen species is in balance with antioxidants.
Mycotoxins, such as deoxynivalenol, aflatoxin, zearalenone and fumonisin can be found in animal feed and can result in nmegative animal performance or illness. Compounds which can manage the levels of mycotoxin, such as via deactivation of the mycotoxin or via binding of the mycotoxin, can be added to the feed to ameliorate these negative effects. Examples of mycotoxin management compounds are Vitafix®, Vitafix Ultra (Nuscience), Mycofix®, Mycofix® Secure, FUMzyme®, Biomin® BBSH, Biomin® MTV (Biomin), Mold-Nil®, Toxy-Nil® and Unike® Plus (Nutriad).
Embodiments of the Invention
Preferred embodiments of the invention are described in the set of items below.
1. Use of a combination of a polypeptide having alpha-galactosidase activity and a polypeptide having xylanase activity in an animal feed or animal feed additive, wherein the polypeptide having alpha-galactosidase activity is a GH36 alpha-galactosidase and wherein the polypeptide having xylanase activity is a GH30 xylanase.
2. The use of a combination according to embodiment 1 wherein the polypeptide having alpha- galactosidase activity is a GH36 alpha-galactosidase selected from the group consisting of a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide having at least 90% sequence identity to SEQ ID NO: 9; g) a polypeptide having at least 90% sequence identity to SEQ ID NO: 11 ; h) a polypeptide having at least 90% sequence identity to SEQ ID NO: 12; i) a polypeptide having at least 90% sequence identity to SEQ ID NO: 14; j) a polypeptide having at least 90% sequence identity to SEQ ID NO: 15; k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16;
L) a polypeptide having at least 90% sequence identity to SEQ ID NO: 18; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; n) a polypeptide having at least 90% sequence identity to SEQ ID NO: 21 ; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO: 22.
3. The use of a combination according to embodiment 1 or 2 wherein the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; q) a polypeptide having at least 90% sequence identity to SEQ ID NO: 24; r) a polypeptide having at least 90% sequence identity to SEQ ID NO: 25; s) a polypeptide having at least 90% sequence identity to SEQ ID NO: 26; t) a polypeptide having at least 90% sequence identity to SEQ ID NO: 27; u) a polypeptide having at least 90% sequence identity to SEQ ID NO: 28; v) a polypeptide having at least 90% sequence identity to SEQ ID NO: 29; w) a polypeptide having at least 90% sequence identity to SEQ ID NO: 30; x) a polypeptide having at least 90% sequence identity to SEQ ID NO: 31 ; y) a polypeptide having at least 90% sequence identity to SEQ ID NO: 32; z) a polypeptide having at least 90% sequence identity to SEQ ID NO: 33; aa) a polypeptide having at least 90% sequence identity to SEQ ID NO: 34; bb) a polypeptide having at least 90% sequence identity to SEQ ID NO: 35; cc) a polypeptide having at least 90% sequence identity to SEQ ID NO: 36; dd) a polypeptide having at least 90% sequence identity to SEQ ID NO: 37; ee) a polypeptide having at least 90% sequence identity to SEQ ID NO: 38; ft) a polypeptide having at least 90% sequence identity to SEQ ID NO: 39; and gg) a polypeptide having at least 90% sequence identity to SEQ ID NO: 40. The use of a combination according to any of embodiments 1 to 3 wherein the polypeptide having alpha-galactosidase activity is a GH36 alpha-galactosidase selected from the group consisting of a) a polypeptide obtained or obtainable from Bacillus deramificans having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide obtained or obtainable from Bacillus deramificans having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide obtained or obtainable from Bacillus acidopullulyticus having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide obtained or obtainable from Bacillus acidopullulyticus having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide obtained or obtainable from Anoxybacillus bogrovensis having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide obtained or obtainable from Anoxybacillus bogrovensis having at least 90% sequence identity to SEQ ID NO: 9; g) a polypeptide obtained or obtainable from Aspergillus sydowii having at least 90% sequence identity to SEQ ID NO: 11 ; h) a polypeptide obtained or obtainable from Aspergillus sydowii having at least 90% sequence identity to SEQ ID NO: 12; i) a polypeptide obtained or obtainable from Bacillus sp-19140 having at least 90% sequence identity to SEQ ID NO: 14; j) a polypeptide obtained or obtainable from Bacillus sp-19140 having at least 90% sequence identity to SEQ ID NO: 15; k) a polypeptide obtained or obtainable from Aspergillus niger having at least 90% sequence identity to SEQ ID NO: 16; L) a polypeptide obtained or obtainable from Aspergillus puniceus having at least 90% sequence identity to SEQ ID NO: 18; m)a polypeptide obtained or obtainable from Aspergillus puniceus having at least 90% sequence identity to SEQ ID NO: 19; n) a polypeptide obtained or obtainable from PeniciIHum pseudopulvillorum having at least 90% sequence identity to SEQ ID NO: 21 ; and o) a polypeptide obtained or obtainable from PeniciIHum pseudopulvillorum having at least 90% sequence identity to SEQ ID NO: 22.
5. The use of a combination according to any of embodiments 1 to 4 wherein the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide obtained or obtainable from Bacillus subtilis having at least 90% sequence identity to SEQ ID NO: 23; q) a polypeptide obtained or obtainable from Bacillus amyloliquefaciens having at least 90% sequence identity to SEQ ID NO: 24; r) a polypeptide obtained or obtainable from Bacillus licheniformis having at least 90% sequence identity to SEQ ID NO: 25; s) a polypeptide obtained or obtainable Bacillus subtilis having at least 90% sequence identity to SEQ ID NO: 26; t) a polypeptide obtained or obtainable from Paenibacillus pabuli having at least 90% sequence identity to SEQ ID NO: 27; u) a polypeptide obtained or obtainable from Bacillus amyloliquefaciens having at least 90% sequence identity to SEQ ID NO: 28; v) a polypeptide obtained or obtainable from Bacillus subtilis having at least 90% sequence identity to SEQ ID NO: 29; w) a polypeptide obtained or obtainable from Clostridium acetobutylicum having at least 90% sequence identity to SEQ ID NO: 30; x) a polypeptide obtained or obtainable from Pseudoalteromonas tetraodonis having at least 90% sequence identity to SEQ ID NO: 31 ; y) a polypeptide obtained or obtainable from Paenibacillus sp-19179 having at least 90% sequence identity to SEQ ID NO: 32; z) a polypeptide obtained or obtainable from Pectobacterium carotovorum having at least 90% sequence identity to SEQ ID NO: 33; aa) a polypeptide obtained or obtainable from Ruminococcus sp. having at least 90% sequence identity to SEQ ID NO: 34; bb) a polypeptide obtained or obtainable from Streptomyces sp-62627 having at least 90% sequence identity to SEQ ID NO: 35; cc) a polypeptide obtained or obtainable from Clostridium saccharoperbutylacetonicum having at least 90% sequence identity to SEQ ID NO: 36; dd) a polypeptide obtained or obtainable from Paenibacillus panacisoli having at least 90% sequence identity to SEQ ID NO: 37; ee) a polypeptide obtained or obtainable from Human Stool metagenome having at least 90% sequence identity to SEQ ID NO: 38; ff) a polypeptide obtained or obtainable from Vibrio rhizosphaerae having at least 90% sequence identity to SEQ ID NO: 39; and gg) a polypeptide obtained or obtainable from Bacillus sp-18423 having at least 90% sequence identity to SEQ ID NO: 40.
6. The use according to any of embodiments 1 to 5, wherein the polypeptide having alpha- galactosidase activity is a polypeptide selected from a polypetide as defined by a), b), c), d), e), f), k), m), and o).
7. The use according to any of embodiments 1 to 6, wherein the polypeptide having alpha- galactosidase activity is a polypeptide selected from a polypetide as defined by k), m), and o).
8. The use according to any of embodiments 1 to 7, wherein the polypeptide having alpha- galactosidase activity is a polypeptide selected from a polypetide as defined by k).
9. The use according to any of embodiments 1 to 8 wherein the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p), v) and gg).
10. The use according to any of embodiments 1 to 9 wherein the polypeptide having xylanase activity is a GH30 xylanase as defined by p).
11. An animal feed additive comprising a polypeptide having alpha-galactosidase activity and a polypeptide having xylanase activity; wherein the polypeptide having alpha-galactosidase activity is a GH36 alpha-galactosidase and wherein the polypeptide having xylanase activity is a GH30 xylanase.
12. The animal feed additive according to embodiment 11 , wherein the polypeptide having alpha-galactosidase activity is a GH36 alpha-galactosidase selected from the group consisting of a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide having at least 90% sequence identity to SEQ ID NO: 9; g) a polypeptide having at least 90% sequence identity to SEQ ID NO: 11 ; h) a polypeptide having at least 90% sequence identity to SEQ ID NO: 12; i) a polypeptide having at least 90% sequence identity to SEQ ID NO: 14; j) a polypeptide having at least 90% sequence identity to SEQ ID NO: 15; k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16;
L) a polypeptide having at least 90% sequence identity to SEQ ID NO: 18; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; n) a polypeptide having at least 90% sequence identity to SEQ ID NO: 21 ; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO: 22 and wherein the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; q) a polypeptide having at least 90% sequence identity to SEQ ID NO: 24; r) a polypeptide having at least 90% sequence identity to SEQ ID NO: 25; s) a polypeptide having at least 90% sequence identity to SEQ ID NO: 26; t) a polypeptide having at least 90% sequence identity to SEQ ID NO: 27; u) a polypeptide having at least 90% sequence identity to SEQ ID NO: 28; v) a polypeptide having at least 90% sequence identity to SEQ ID NO: 29; w) a polypeptide having at least 90% sequence identity to SEQ ID NO: 30; x) a polypeptide having at least 90% sequence identity to SEQ ID NO: 31 ; y) a polypeptide having at least 90% sequence identity to SEQ ID NO: 32; z) a polypeptide having at least 90% sequence identity to SEQ ID NO: 33; aa)a polypeptide having at least 90% sequence identity to SEQ ID NO: 34; bb)a polypeptide having at least 90% sequence identity to SEQ ID NO: 35; cc) a polypeptide having at least 90% sequence identity to SEQ ID NO: 36; dd)a polypeptide having at least 90% sequence identity to SEQ ID NO: 37; ee)a polypeptide having at least 90% sequence identity to SEQ ID NO: 38; ff) a polypeptide having at least 90% sequence identity to SEQ ID NO: 39; and gg)a polypeptide having at least 90% sequence identity to SEQ ID NO: 40. The animal feed additive according to embodiments 11 or 12, wherein the polypeptide having alpha-galactosidase activity is a polypeptide selected from the group consisting of a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide having at least 90% sequence identity to SEQ ID NO: 9; k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; n) a polypeptide having at least 90% sequence identity to SEQ ID NO: 21 ; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO: 22 wherein the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; и) a polypeptide having at least 90% sequence identity to SEQ ID NO: 29; and ff) a polypeptide having at least 90% sequence identity to SEQ ID NO: 40. The animal feed additive according to any of embodiments 11 to 13, wherein the polypeptide having alpha-galactosidase activity is a polypeptide selected from the group consisting of к) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO: 22 wherein the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; u) a polypeptide having at least 90% sequence identity to SEQ ID NO: 29; and ff) a polypeptide having at least 90% sequence identity to SEQ ID NO: 40. The animal feed additive according to any of embodiments 11 to 14, wherein the polypeptide having alpha-galactosidase activity is k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; and wherein the polypeptide having xylanase activity p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23. The animal feed additive according to any of embodiments 11 to 15 further comprising one or more additional components selected from the group consisting of: one or more vitamins; one or more minerals; one or more amino acids; one or more phytogenies; one or more prebiotics; one or more organic acids; and one or more other feed ingredients
17. The animal feed additive according to any of embodiments 11 to 16 wherein the amount of alpha-galactosidase and xylanase increases the Average Metabolizable Energy of plant- based diet.
18. A method of improving the Average Metabolizable Energy of plant-based diet in a monogastric animal comprising administering an animal feed additive as defined in any of embodiments 11-16 or the combination as defined in any of embodiments 1 to 10.
19. A composition comprising the combination as defined in any of embodiments 1 to 10.
20. The composition according to embodiment 19 in the form of a granule.
21. The composition according to embodiment 19 in the form of a liquid formulation.
22. An animal feed comprising the combination as defined in any of embodiments 1 to 10, or the animal feed additive as defined in any of embodiments 11 to 17, and further comprising plant-based material.
23. A method of releasing starch from plant-based material in animal feed, comprising treating plant based material with the combination as defined in any of embodiments 1 to 10, or the animal feed additive as defined in any of embodiments 11 to 17 or the composition as defined in any of embodiments 19 to 21.
24. A method for improving the nutritional value of an animal feed, said feed comprising plant based material comprising adding to the feed comprising plant based material the combination as defined in any of embodiments 1 to 10, or the animal feed additive as defined in any of embodiments 11 to 17 or the composition as defined in any of embodiments 19 to 21.
25. The animal feed additive according to embodiments 11 to 17 wherein the additive is for feed for monogastric animals. 26. The animal feed additive according to embodiment 25 wherein the monogastric animal is selected from the group consisting of 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).
27. A method of feeding an animal comprising adding the combination as defined in any of embodiments 1 to 10, or the animal feed additive as defined in any of embodiments 11 to 17 or the composition as defined in any of embodiments 19 to 21 to the animal feed.
28. The animal feed according to embodiment 22 comprising maize, soybean meal or both.
29. The animal feed according to embodiment 22 comprising as a primary protein source maize or soybean meal or a combination thereof.
30. A composition according to embodiment 19 and further comprising a formulating agent.
31 . The composition of embodiment 30, wherein the formulating agent comprises one or more of the following compounds: 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, maltodextrin, cyclodextrin, wheat, PVA, acetate, phosphate and cellulose.
32. The composition according to embodiment 19, further comprising one or more additional enzymes.
33. The composition of embodiment 32, wherein the one or more additional enzymes is selected from the group consisting of acetyl xylan esterase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha- galactosidase, beta-galactosidase, beta-glucanase, beta-glucosidase, lipase, glucan 1 ,4- a-glucosidase, glucan 1 ,4-alpha-maltohydrolase, lysophospholipase, lysozyme, mannanase, alpha-mannosidase, beta-mannosidase, phytase, phospholipase A1, phospholipase A2, phospholipase C, phospholipase D, protease, pullulanase, pectinase, pectin lyase, xylanase, beta-xylosidase, or any combination thereof.
34. The composition of any of embodiments 30 to 33, further comprising one or more microbes. 35. The composition of embodiment 34, wherein the one or more microbes is selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium sp., Carnobacterium sp., Clostridium butyricum, Clostridium sp., Enterococcus faecium, Enterococcus sp., Lactobacillus sp., Lactobacillus acidophilus, Lactobacillus farciminus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, Lactococcus lactis, Lactococcus sp., Leuconostoc sp., Megasphaera elsdenii, Megasphaera sp., Pediococsus acidilactici, Pediococcus sp., Propionibacterium thoenii, Propionibacterium sp. and Streptococcus sp. or any combination thereof.
36. The composition of any of embodiments 30 to 35, further comprising plant based material.
37. The composition of embodiment 36, wherein the plant based material is from the sub-family Panicoideae.
38. The composition of embodiment 37, wherein the plant based material from the sub-family Panicoideae is maize, corn, sorghum, switchgrass, millet, pearl millet, foxtail millet or in a processed form such as milled corn, milled maize, defatted maize, defatted destarched maize, milled sorghum, milled switchgrass, milled millet, milled foxtail millet, milled pearl millet, or any combination thereof.
39. The composition of embodiments 36 to 38, wherein the plant based material from the subfamily Panicoideae is from the seed fraction (such as endosperm and/or husk) of the plant.
40. A granule comprising the combination of any of embodiments 1 to 10 and a formulating agent.
41. The granule of embodiment 40, wherein the one or more formulating agents is 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, preferably selected from the list consisting of 1 , 2-propylene glycol, 1 , 3-propylene glycol, sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate. 42. The granule of any of embodiments 40 to 41 , wherein the granule comprises a core particle and one or more coatings
43. The granule of embodiment 42, wherein the coating comprises salt and/or wax and/or flour.
44. The granule of any of embodiments 40 to 43 further comprising one or more additional enzymes.
45. The granule of embodiments 44, wherein the one or more additional enzymes is selected from the group consisting of acetyl xylan esterase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha- galactosidase, beta-galactosidase, beta-glucanase, beta-glucosidase, lipase, glucan 1 ,4- a-glucosidase, glucan 1 ,4-alpha-maltohydrolase, lysophospholipase, lysozyme, mannanase, alpha-mannosidase, beta-mannosidase, phytase, phospholipase A1, phospholipase A2, phospholipase C, phospholipase D, protease, pullulanase, pectinase, pectin lyase, xylanase, beta-xylosidase, or any combination thereof.
The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.
Examples
Example 1: Proof of concept standard protocol
Cage broiler studies are designed for the evaluation of the combination of soy carbohydrate degrading enzymes on growth performance and apparent metabolisable energy corrected to zero N retention.
The present describes the mandatory conditions/specifications for each internal and external trial.
Dosage:
Figure imgf000060_0001
Figure imgf000061_0001
A: NC; B: triple high - comprising SEQ ID NO:16 - 20ppm EP each : C: triple low - comprising SEQ ID NO:16 - 5ppm EP each; D: single high - comprising SEQ ID NO:16 - 20ppm EP; E: single low - comprising SEQ ID NO:16 - 5ppm EP
Feed composition
Diet with 55-65 % corn and 25-35 % SBM (see Basal diet formulation)
• The diets formulated to contain approx. 210 g crude protein and 12.7 MJ/kg ME for the experimental period (day 8-28)
• Vitamin levels: industrial practice levels
• Ronozyme HiPhos at 1000 FYT/kg feed
• Use of coccidiostat in all diets: according to local practice
Feeding phase:
• pre-experimental diet (day 0-8)
• experimental diet with enzyme supplementation (day 8-28)
• Feed composition provided per feeding phase
• Feed distribution: pellets or mash, according to the institute selected and available
• Pelleting process conditions: 70°C, 30 sec, 3 x 25
• Feed storage conditions: 4°C until distribution
Addition of testing products:
• Based on the quantity of feed, the enzyme needed ready to be sprayed directly onto pellet or mash diet is prepared.
• Final volume of the product solution is expected to be: 300 to 500 ml for ~200 kg diet.
• To determine apparent total tract metabolisability of nutrients, indigestible marker should be included in the experimental diets (i.e: Titanium dioxide T1O2, acid-insoluble ash or chromium oxide). Alternatively a total collection method may be used.
Animal and housing
• Day-old male broiler chickens (Ross 308/708, or Cobb 500) to be supplied by a local commercial hatchery
• Environmental conditions to be described • Battery cage dimensions to be specified
• Birds should be kept in cages and fed pre-experimental diet until 8 day of age
• On d8 birds in the bottom 10% and top 10% of weight should be removed from the experiment
• At 8 day of age, birds are randomly allocated (but weighed) into groups of 6 to 8 birds (according to the size of the cage) to the n dietary treatments and housed in battery cages
• Each treatment is replicated with 8 to 12 groups
• At day 8, birds are fed ad libitum with their respective experimental diets
• Water is available ad libitum: fresh and clear water is provided daily
Productive performance.
• Body weight and feed intake /cage are recorded at days 8, 15, 21 and 28.
• Weight gains, feed intakes and feed conversion ratios of each cage of birds will be determined at 8, 15, 21 and 28 day of the trial, and calculated for each feeding period and for the whole experiment.
• General health records (e.g. diarrhoea, respiratory problems).
• Mortality and weight of dead birds are recorded as it occurs and percentage of mortality calculated.
• Daily records of the maximum and minimum temperatures, relative humidity and all other routine activities.
• Unusual incidents/adverse events. Any abrupt changes in the weather, disease outbreaks, power failures, feed/water blockages, frozen/burst pipes, etc. will be noted.
Chemical analysis
Figure imgf000062_0001
• The analyses of the nutrient contents in feed, excreta and digesta samples are performed according to the standard methods
• Nitrogen (N) is determined using automated nitrogen analyser (LECO) according to Dumas combustion method or using Kjeldahl procedure (protein contents are calculated using the formula: 6.25 x N). • Gross energy (GE) is determined using an adiabatic bomb calorimeter standardized with benzoic acid.
Calculations
The N-corrected apparent metabolizable energy (AMEn) values are calculated according to the following equation:
AMEn (MJ/kg DM) = [(FE -EE) - 8.22 x N ]/ FI
Where: FE = total feed energy (kcal); EE = total excreta energy (kcal)
FI = total feed intake (g); N = nitrogen retention (kg)
Procedures of sampling
Excreta total collection (day 21 to day 24)
• Birds are adapted ad libitum to the experimental feed (day 8 to 21)
• Day 21 : installation of the collection device of excreta,
• The total amount of feed consumed during the excreta collection period is determined
• The excreta are collected daily from day 21 to day 24.
• The excreta (n replicates of 6 to 8 birds per treatment) from three days are pooled per group and stored frozen (at -20°C) each day immediately after collection in closed plastic bags
• The excreta collection is done at the same hour each day, during the balance period
• Excreta samples are collected treatment after treatment and in the same order and timing
• A total collection method may be used or, after sampling marker analysis may be conducted as per the institutes protocols.
Sample collection, handling, storage
• After frozen and thawing, the total excreta of each group are homogenized, freeze- dried, and ground (0, 5 mm) for chemical analysis
• Samples are stored in closed plastic tubes at room temperature
Example 2: Chick progress Report
Average Metabolizable Energy (MJ/kg DM, relative Negative Control %)
Figure imgf000063_0001
Figure imgf000064_0001
Conclusion: The combination of the xylanase and the alpha-galactosidase surprisingly increases the Average Metabolizable Energy (AME) to a more than additive extent. As known in the art, a 2% increase in AME translates to commercially relevant savings to the farmer. At doses of 5 or 10 ppm of xylanase, depending on the study, there is virtually no increase in AME. Adding alpha-galactosidase to the 5 ppm of xylanase, slight but signficants increases in AME is achieved. At higher doses of xylanase, the addition of alpha-galactosidase increases the AME dramatically to up to as much as 5.7%.
Example 3: Animal feed and animal feed additives
Granule
The granule is prepared by granulating a xylanase variant of the invention with a filler such as sodium sulfate, magnesium sulfate, calcium carbonate and/or cellulose and then optionally coating the granule with a wax coating (e.g. hydrogenated palm oil) or a salt coating (e. g. sodium sulfate and/or magnesium sulfate).
Alternatively, granule is prepared by absorbing a liquid solution of a xylanase variant of the invention onto an inert core and then optionally coating the granule with a wax coating (e.g. hydrogenated palm oil) or a salt coating (e. g. sodium sulfate and/or magnesium sulfate).
Animal Feed Additive
A premix formulation of a xylanase variant of the invention containing 0.01 g to 10g enzyme protein per kilo of premix (optionally formulated as a coated granule) is added to the following premix:
5000000 IE Vitamin A
1000000 IE Vitamin D3
13333 mg Vitamin E
1000 mg Vitamin K3
750 mg Vitamin B1 2500 mg Vitamin B2
1500 mg Vitamin B6
7666 meg Vitamin B12
12333 mg Niacin
33333 meg Biotin
300 mg Folic Acid
3000 mg Ca-D-Panthothenate
1666 mg Cu
16666 mg Fe
16666 mg Zn
23333 mg Mn
133 mg Co
66 mg I
66 mg Se
5.8 % Calcium
25 % Sodium
Animal Feed
This is an example of an animal feed (broiler feed) comprising the animal feed additive as described above:
62.55 % Maize
33.8% Soybean meal (50% crude protein)
1.0% Soybean oil
0.2% DL-Methionine
0.22% DCP (dicalcium phosphate)
0.76% CaC03 (calcium carbonate)
0.32% Sand
0.15% NaCI (sodium chloride)
1 % of the above Premix
The ingredients are mixed, and the feed is pelleted at the desired temperature, e.g. 60, 65, 75, 80, 85, 90 or even 95°C.
Liquid Formulation
A liquid formulation of a xylanase variant of the invention comprises 0.1% to 10 w/w enzyme protein, 40-60% glycerol, 0.1 to 0.5% sodium benzoate and water. The liquid formulation is sprayed onto the pelleted animal feed described above. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Claims

1. Use of a combination of a polypeptide having alpha-galactosidase activity and a polypeptide having xylanase activity in an animal feed or animal feed additive, wherein the polypeptide having alpha-galactosidase activity is a GH36 alpha-galactosidase and wherein the polypeptide having xylanase activity is a GH30 xylanase.
2. The use of a combination according to claim 1 wherein the polypeptide having alpha- galactosidase activity is a GH36 alpha-galactosidase selected from the group consisting of a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 2; preferably, a polypeptide obtained or obtainable from Bacillus deramificans having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide having at least 90% sequence identity to SEQ ID NO: 3; preferably, a polypeptide obtained or obtainable from Bacillus deramificans having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide having at least 90% sequence identity to SEQ ID NO: 5; preferably, a polypeptide obtained or obtainable from Bacillus acidopullulyticus having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide having at least 90% sequence identity to SEQ ID NO: 6; preferably, a polypeptide obtained or obtainable from Bacillus acidopullulyticus having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide having at least 90% sequence identity to SEQ ID NO: 8; preferably, a polypeptide obtained or obtainable from Anoxybacillus bogrovensis having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide having at least 90% sequence identity to SEQ ID NO: 9; preferably, a polypeptide obtained or obtainable from Anoxybacillus bogrovensis having at least 90% sequence identity to SEQ ID NO: 9; g) a polypeptide having at least 90% sequence identity to SEQ ID NO: 11 ; preferably, a polypeptide obtained or obtainable from Aspergillus sydowii having at least 90% sequence identity to SEQ ID NO: 11 ; h) a polypeptide having at least 90% sequence identity to SEQ ID NO: 12; preferably, a polypeptide obtained or obtainable from Aspergillus sydowii having at least 90% sequence identity to SEQ ID NO: 12; i) a polypeptide having at least 90% sequence identity to SEQ ID NO: 14; preferably, a polypeptide obtained or obtainable from Bacillus sp-19140 having at least 90% sequence identity to SEQ ID NO: 14; j) a polypeptide having at least 90% sequence identity to SEQ ID NO: 15; preferably, a polypeptide obtained or obtainable from Bacillus sp-19140 having at least 90% sequence identity to SEQ ID NO: 15; k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; preferably, a polypeptide obtained or obtainable from Aspergillus niger having at least 90% sequence identity to SEQ ID NO: 16;
L) a polypeptide having at least 90% sequence identity to SEQ ID NO: 18; preferably, a polypeptide obtained or obtainable from Aspergillus puniceus having at least 90% sequence identity to SEQ ID NO: 18; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; preferably, a polypeptide obtained or obtainable from Aspergillus puniceus having at least 90% sequence identity to SEQ ID NO: 19; n) a polypeptide having at least 90% sequence identity to SEQ ID NO: 21; preferably, a polypeptide obtained or obtainable from PeniciIHum pseudopulvillorum having at least 90% sequence identity to SEQ ID NO: 21 ; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO: 22; preferably, a polypeptide obtained or obtainable from PeniciIHum pseudopulvillorum having at least 90% sequence identity to SEQ ID NO: 22.
3. The use of a combination according to claim 1 or 2 wherein the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; preferably, a polypeptide obtained or obtainable from Bacillus subtilis having at least 90% sequence identity to SEQ ID NO: 23; q) a polypeptide having at least 90% sequence identity to SEQ ID NO: 24; preferably, a polypeptide obtained or obtainable from Bacillus amyloliquefaciens having at least 90% sequence identity to SEQ ID NO: 24; r) a polypeptide having at least 90% sequence identity to SEQ ID NO: 25; preferably, a polypeptide obtained or obtainable from Bacillus licheniformis having at least 90% sequence identity to SEQ ID NO: 25; s) a polypeptide having at least 90% sequence identity to SEQ ID NO: 26; preferably, a polypeptide obtained or obtainable Bacillus subtilis having at least 90% sequence identity to SEQ ID NO: 26; t) a polypeptide having at least 90% sequence identity to SEQ ID NO: 27; preferably, a polypeptide obtained or obtainable from Paenibacillus pabuli having at least 90% sequence identity to SEQ ID NO: 27; u) a polypeptide having at least 90% sequence identity to SEQ ID NO: 28; preferably, a polypeptide obtained or obtainable from Bacillus amyloliquefaciens having at least 90% sequence identity to SEQ ID NO: 28; v) a polypeptide having at least 90% sequence identity to SEQ ID NO: 29; preferably, a polypeptide obtained or obtainable from Bacillus subtilis having at least 90% sequence identity to SEQ ID NO: 29; w) a polypeptide having at least 90% sequence identity to SEQ ID NO: 30; preferably, a polypeptide obtained or obtainable from Clostridium acetobutylicum having at least 90% sequence identity to SEQ ID NO: 30; x) a polypeptide having at least 90% sequence identity to SEQ ID NO: 31 ; preferably, a polypeptide obtained or obtainable from Pseudoalteromonas tetraodonis having at least 90% sequence identity to SEQ ID NO: 31 ; y) a polypeptide having at least 90% sequence identity to SEQ ID NO: 32; preferably, a polypeptide obtained or obtainable from Paenibacillus sp-19179 having at least 90% sequence identity to SEQ ID NO: 32; z) a polypeptide having at least 90% sequence identity to SEQ ID NO: 33; preferably, a polypeptide obtained or obtainable from Pectobacterium carotovorum having at least 90% sequence identity to SEQ ID NO: 33; aa) a polypeptide having at least 90% sequence identity to SEQ ID NO: 34; preferably, a polypeptide obtained or obtainable from Ruminococcus sp. having at least 90% sequence identity to SEQ ID NO: 34; bb) a polypeptide having at least 90% sequence identity to SEQ ID NO: 35; preferably, a polypeptide obtained or obtainable from Streptomyces sp-62627 having at least 90% sequence identity to SEQ ID NO: 35; cc) a polypeptide having at least 90% sequence identity to SEQ ID NO: 36; preferably, a polypeptide obtained or obtainable from Clostridium saccharoperbutylacetonicum having at least 90% sequence identity to SEQ ID NO: 36; dd) a polypeptide having at least 90% sequence identity to SEQ ID NO: 37; preferably, a polypeptide obtained or obtainable from Paenibacillus panacisoli having at least 90% sequence identity to SEQ ID NO: 37; ee) a polypeptide having at least 90% sequence identity to SEQ ID NO: 38; preferably, a polypeptide obtained or obtainable from Human Stool metagenome having at least 90% sequence identity to SEQ ID NO: 38; ff) a polypeptide having at least 90% sequence identity to SEQ ID NO: 39; preferably, a polypeptide obtained or obtainable from Vibrio rhizosphaerae having at least 90% sequence identity to SEQ ID NO: 39; and gg) a polypeptide having at least 90% sequence identity to SEQ ID NO: 40; preferably, a polypeptide obtained or obtainable from Bacillus sp-18423 having at least 90% sequence identity to SEQ ID NO: 40.
4. The use according to any of claims 1 to 3, wherein the polypeptide having alpha- galactosidase activity is a polypeptide selected from a polypetide as defined by a), b), c), d), e), f), k), m), and o); preferably, the polypeptide having alpha-galactosidase activity is a polypeptide selected from a polypetide as defined by k), m), and o); more preferably the polypeptide having alpha-galactosidase activity is a polypeptide selected from a polypetide as defined by k).
5. The use according to any of claims 1 to 4 wherein the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p), v) and gg); preferably, the polypeptide having xylanase activity is a GH30 xylanase as defined by p).
6. An animal feed additive comprising a polypeptide having alpha-galactosidase activity and a polypeptide having xylanase activity; wherein the polypeptide having alpha-galactosidase activity is a GH36 alpha-galactosidase and wherein the polypeptide having xylanase activity is a GH30 xylanase.
7. The animal feed additive according to claim 6, wherein the polypeptide having alpha- galactosidase activity is a GH36 alpha-galactosidase selected from the group consisting of a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide having at least 90% sequence identity to SEQ ID NO: 9; g) a polypeptide having at least 90% sequence identity to SEQ ID NO: 11 ; h) a polypeptide having at least 90% sequence identity to SEQ ID NO: 12; i) a polypeptide having at least 90% sequence identity to SEQ ID NO: 14; j) a polypeptide having at least 90% sequence identity to SEQ ID NO: 15; k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16;
L) a polypeptide having at least 90% sequence identity to SEQ ID NO: 18; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; n) a polypeptide having at least 90% sequence identity to SEQ ID NO: 21 ; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO: 22; and wherein the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; q) a polypeptide having at least 90% sequence identity to SEQ ID NO: 24; r) a polypeptide having at least 90% sequence identity to SEQ ID NO: 25; s) a polypeptide having at least 90% sequence identity to SEQ ID NO: 26; t) a polypeptide having at least 90% sequence identity to SEQ ID NO: 27; u) a polypeptide having at least 90% sequence identity to SEQ ID NO: 28; v) a polypeptide having at least 90% sequence identity to SEQ ID NO: 29; w) a polypeptide having at least 90% sequence identity to SEQ ID NO: 30; x) a polypeptide having at least 90% sequence identity to SEQ ID NO: 31 ; y) a polypeptide having at least 90% sequence identity to SEQ ID NO: 32; z) a polypeptide having at least 90% sequence identity to SEQ ID NO: 33; aa) a polypeptide having at least 90% sequence identity to SEQ ID NO: 34; bb) a polypeptide having at least 90% sequence identity to SEQ ID NO: 35; cc) a polypeptide having at least 90% sequence identity to SEQ ID NO: 36; dd) a polypeptide having at least 90% sequence identity to SEQ ID NO: 37; ee) a polypeptide having at least 90% sequence identity to SEQ ID NO: 38; ff) a polypeptide having at least 90% sequence identity to SEQ ID NO: 39; and gg) a polypeptide having at least 90% sequence identity to SEQ ID NO: 40.
8. The animal feed additive according to claim 6 or 7, wherein the polypeptide having alpha- galactosidase activity is a polypeptide selected from the group consisting of a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 2; b) a polypeptide having at least 90% sequence identity to, SEQ ID NO: 3; c) a polypeptide having at least 90% sequence identity to SEQ ID NO: 5; d) a polypeptide having at least 90% sequence identity to SEQ ID NO: 6; e) a polypeptide having at least 90% sequence identity to SEQ ID NO: 8; f) a polypeptide having at least 90% sequence identity to SEQ ID NO: 9; k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; n) a polypeptide having at least 90% sequence identity to SEQ ID NO: 21 ; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO: 22 wherein the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; u) a polypeptide having at least 90% sequence identity to SEQ ID NO: 29; and gg) a polypeptide having at least 90% sequence identity to SEQ ID NO: 40.
9. The animal feed additive according to any of claims 6 to 8, wherein the polypeptide having alpha-galactosidase activity is a polypeptide selected from the group consisting of k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; m) a polypeptide having at least 90% sequence identity to SEQ ID NO: 19; and o) a polypeptide having at least 90% sequence identity to SEQ ID NO: 22 wherein the polypeptide having xylanase activity is a GH30 xylanase selected from the group consisting of p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23; u) a polypeptide having at least 90% sequence identity to SEQ ID NO: 29; and gg) a polypeptide having at least 90% sequence identity to SEQ ID NO: 40.
10. The animal feed additive according to any of claims 6 to 9, wherein the polypeptide having alpha-galactosidase activity is k) a polypeptide having at least 90% sequence identity to SEQ ID NO: 16; and wherein the polypeptide having xylanase activity p) a polypeptide having at least 90% sequence identity to SEQ ID NO: 23.
11 . The animal feed additive according to any of claims 6 to 10 further comprising one or more additional components selected from the group consisting of: one or more vitamins; one or more minerals; one or more amino acids; one or more phytogenies; one or more prebiotics; one or more organic acids; and one or more other feed ingredients.
12. The animal feed additive according to claims 6 to 11 wherein the additive is for feed for monogastric animals; preferably, the monogastric animal is selected from the group consisting of 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).
13. A method of improving the Average Metabolizable Energy of plant-based diet in a monogastric animal comprising administering an animal feed additive as defined in any of claims 6-12 or the combination as defined in any of claims 1 to 5.
14. A composition comprising the combination as defined in any of claims 1 to 5.
15. The composition according to claim 14 in the form of a granule or a liquid formulation.
16. An animal feed comprising the combination as defined in any of claims 1 to 5, or the animal feed additive as defined in any of claims 6 to 12, and further comprising plant-based material.
17. The animal feed according to claim 16 comprising maize, soybean meal or both; or comprising as a primary protein source maize or soybean meal or a combination thereof.
18. A method of releasing starch from plant based material in animal feed, comprising treating plant based material with the combination as defined in any of claims 1 to 5, or the animal feed additive as defined in any of claims 6 to 12 or the composition as defined in any of claims 14 to 15.
19. A method for improving the nutritional value of an animal feed, said feed comprising plant based material comprising adding to the feed comprising plant based material, the combination as defined in any of claims 1 to 5, or the animal feed additive as defined in any of claims 6 to 12 or the composition as defined in any of claims 14 to 15.
20. A method of feeding an animal comprising adding the combination as defined in any of claims 1 to 5, or the animal feed additive as defined in any of claims 6 to 12, or the composition as defined in any of claims 14 to 15 to the animal feed.
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