Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/189—Enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
  • A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
  • A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
  • A23K10/38—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material from distillers' or brewers' waste
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
• • 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)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
  • C12Y301/03008—3-Phytase (3.1.3.8)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
  • C12Y301/03026—4-Phytase (3.1.3.26), i.e. 6-phytase

Definitions

• FIELD The field pertains to ruminant diets containing no or substantially no or decreased inorganic phosphate as well as engineered phytase polypeptides and uses of the same for enhancing dairy production in ruminant animals.
• BACKGROUND Ruminant livestock accounts for up to 70% of the phosphorus (P) waste produced by farmed animals (Tamminga and Verstegen, 1992). Reducing P excretion from dairy cattle continues to be a major focus to minimize the negative impacts of excess P on the environment, especially on aquatic ecosystems (Sharpley et al., 1994; NRC, 2001). Optimizing dietary P content and improving P utilization are key strategies for reducing P excretion in dairy cows (Valk et al., 2000).
• NB42148-WO-PCT intestine (Raun et al., 1956; Yanke et al., 1998; Guyton et al., 2003).
• Morse et al. (1992) reported that phytate-P (PP) disappearance from a range of cereal and oilseed meal concentrates incubated with ruminal fluid was greater than 99% in vitro, and that apparent total tract digestibility (ATTD) of PP was 94 to 99% in lactating dairy cows.
• Clark et al. (1986) reported ATTD of PP values higher than 95% in high producing dairy cows.
• the neutral pH of the rumen that is typically pH 6.0 to 7.0 (Winter et al., 2015; Kim et al., 2018) may not be optimal for ruminal microbes producing cysteine phytases that exhibit optimum activity at pH 4.5 (Yanke et al., 1998; Puhl et al., 2008).
• Exogenous microbial phytases mostly of bacterial origin, are widely used as feed additives in commercial poultry and swine diets for improving P digestibility and utilization via their hydrolysis of phytate (Selle and Ravindran, 2007; Humer et al., 2015).
• NB42148-WO-PCT improved protein utilization during lactation compared to exogenous phytase-free diets that are supplemented with sources of inorganic phosphate.
• SUMMARY Provided herein, inter alia, are ruminant animal diets containing phytases which are free or substantially free of exogenously added inorganic phosphate or contain substantially reduced exogenously added inorganic phosphate. When fed to ruminant animals (for example, lactating ruminant animals), these diets ensure improved phosphorous and/or protein utilization (manifested by, for example, improved digestibility, decreased excretion of crude protein, and/ improved milk protein content and/or yield) compared to diets which contain inorganic phosphate supplementation.
• phytase polypeptide or a fragment thereof comprises at least 82% sequence identity with the amino acid sequence set forth in SEQ ID NO:1.
• improving protein utilization comprises a) improved digestibility and/or decreased excretion of crude protein; and/or b) improved milk protein content and protein yield.
• improved phosphorous utilization comprises improved digestibility and/or decreased excretion of total phosphorus and/or phytate bound phosphorous.
• the method further improves calcium digestibility.
• the lactating ruminant animal is selected from the group consisting of cattle, young calves, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, reindeer, caribou, camels, alpacas, llamas, antelope, pronghorn and nilgai.
• the phytase polypeptide or a fragment thereof comprising phytase activity is not coated. In some embodiments of any of the embodiments disclosed herein, the phytase polypeptide or a fragment thereof comprising phytase activity is formulated for bypassing the rumen. In some embodiments of any of the embodiments disclosed herein, the phytase polypeptide or a fragment thereof comprising phytase activity is administered in combination with a feed. In some embodiments, the feed is selected from the Attorney Docket No. NB42148-WO-PCT group consisting of total mixed ration (TMR), compound feed, mineral premix.
• TMR total mixed ration
• the mineral premix is administered as a licking block.
• the phytase polypeptide or a fragment thereof comprising phytase activity is administered in a water line.
• the method further comprises administering at least one additional enzyme to the animal.
• the phytase polypeptide is administered in conjunction with a diet having low phosphorous content.
• the phytase polypeptide or a fragment thereof comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:10, SEQ ID NO:11,
• a method for decreasing phosphate and/or nitrogen excretion in the feces of a lactating ruminant animal comprising administering to the animal a phytase polypeptide or a fragment thereof comprising phytase activity.
• the phytase polypeptide or a fragment thereof comprises at least 82% sequence identity with the amino acid sequence set forth in SEQ ID NO:1.
• the lactating ruminant animal is selected from the group consisting of cattle, young calves, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, reindeer, caribou, camels, alpacas, llamas, antelope, pronghorn and nilgai.
• the phytase polypeptide or a fragment thereof comprising phytase activity is not coated.
• the phytase polypeptide or a fragment thereof comprising phytase activity is formulated for bypassing the rumen.
• the phytase polypeptide or a fragment thereof comprising phytase activity is administered in combination with a feed.
• the feed is selected from the group consisting of total mixed ration (TMR), compound feed, mineral premix.
• the mineral premix is administered as a licking block.
• the phytase polypeptide or a fragment thereof comprising phytase activity is administered in a water line.
• the method further comprises administering at least one additional enzyme to the animal.
• the phytase polypeptide is administered in conjunction with a diet having low phosphorous content.
• the phytase polypeptide or a fragment thereof comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:10, SEQ ID NO:11,
• a method for degrading phytate in the abomasum of a ruminant animal comprising administering to the animal a phytase polypeptide or a fragment thereof comprising at least 82% sequence identity of the amino acid sequence set forth in SEQ ID NO:1.
• the phytase polypeptide or a fragment thereof degrades phytate in the abomasum to a greater extent as compared to a phytase polypeptide or a fragment thereof that does not comprise at least 82% sequence identity of the amino acid sequence set forth in SEQ ID NO:1.
• the lactating ruminant animal is selected from the group consisting of cattle, young calves, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, reindeer, caribou, camels, alpacas, llamas, antelope, pronghorn and nilgai.
• the phytase polypeptide or a fragment thereof comprising phytase activity is not coated.
• the phytase polypeptide or a fragment thereof comprising phytase activity is administered in combination with a feed.
• the feed is selected from the group consisting of total mixed ration (TMR), compound feed, mineral premix.
• the feed comprises one or more cereal byproducts of a distillation process.
• the phytase polypeptide or a fragment thereof comprising phytase activity is used during a saccharification and/or fermentation reaction prior to the distillation process. In some Attorney Docket No.
• the feed comprises one or more of corn gluten meal, Distillers Dried Grains with Solubles (DDGS), corn based Distillers Dried Grains with Solubles (cDDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, or citrus pulp.
• the mineral premix is administered as a licking block.
• the phytase polypeptide or a fragment thereof comprising phytase activity is administered in a water line.
• the method further comprises administering at least one additional enzyme to the animal.
• the phytase polypeptide is administered in conjunction with a diet having low phosphorous content.
• the phytase polypeptide or a fragment thereof comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:10, SEQ ID NO:11,
• FIG. 1 is a graph depicting the in vitro evaluation showing percentage (%) of undegraded IP6 during rumen (pH 6.2) and abomasum (pH 2.0) simulation.
• next generation biosynthetic bacterial 6-phytases in lactating ruminant diets can decrease or eliminate the need to supplement the diet with one or more sources of inorganic phosphate and/or can substantially reduce the need to supplement the diet with one or more sources of inorganic phosphate. Consequently, use of the phytase supplemented inorganic phosphate-free diets disclosed herein provides both an economic advantage in the form of decreased costs of feed as well as a significant environmental benefit due to decreased phosphate pollution as a byproduct of large-scale dairy production. All patents, patent applications, and publications cited are incorporated herein by reference in their entirety. In this disclosure, many terms and abbreviations are used.
• a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
• the term “and/or” and “or” are used interchangeably herein and refer to a specific disclosure of each of the two specified features or components with or without the other.
• the term “and/or” as used in a phrase such “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” alone.
• the term “and/or” as used a phrase such as “A, B and/or C” is intended to encompass each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). Words using the singular include the plural, and vice versa.
• range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the embodiments described herein. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range.
• description of a range, such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 2, from 1 to 3, from 1 to 4 and from 1 to 5, from 2 to 3, from 2 to 4, from 2 to 5, from 2 to 6, from 3 to 4, from 3 to 5, from 3 to 6, etc. as well as individual numbers within that range, for example, 1, 2, 3, 4, 5 and 6. This applies regardless of the breadth of the range.
• phytase (myo-inositol hexakisphosphate phosphohydrolase) refers to a class of phosphatase enzymes that catalyzes the hydrolysis of phytic acid (myo-inositol hexakisphosphate or IP6) – an indigestible, organic form of phosphorus that is found in grains and oil seeds – and releases a usable form of inorganic phosphorus.
• ruminants or “ruminant animals” as used herein refers to mammals, both males and females, that are able to acquire nutrients from plant-based food through fermentation in a specialized stomach chamber prior to digestion, principally through bacterial actions.
• the process typically requires regurgitation of fermented ingesta (known as cud) and chewing it again.
• the process of rechewing the cud to further break down plant matter and stimulate digestion is called “rumination”'.
• the primary difference between ruminant animals and non- ruminant animals is that ruminant animals have a three or four-chambered stomach.
• the group includes, among others, deer, antelopes, buffalo, cattle, sheep, camel, and goat.
• the term “lactating ruminant” as used herein refers to a ruminant animal which is capable of producing milk post-parturition.
• Attorney Docket No. NB42148-WO-PCT The term “dairy ruminant” as used herein refers to a ruminant animal, whose milk is used for commercial purposes including the period that the animal is not giving milk.
• feed an “animal feed,” or “diet” are used interchangeably herein to mean any natural or artificial diet, meal or the like or components of such meals intended or suitable for being eaten, taken in, digested, by a non-human animal, respectively.
• feed is used with reference to products that are fed to animals in the rearing of livestock.
• a “feed additive” as used herein refers to one or more ingredients, products of substances (e.g., cells), used alone or together, in nutrition (e.g., to improve the quality of a food (e.g., an animal feed), to improve an animal’s performance and/or health, and/or to enhance digestibility of a food or materials within a food.
• the term "food” is used in a broad sense - and covers food and food products in any form for humans as well as food for animals (i.e. a feed).
• the food or feed may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration.
• the enzymes mentioned herein may be used as - or in the preparation or production of - a food or feed substance.
• the term "food or feed ingredient” includes a formulation, which is or can be added to foods or foodstuffs and includes formulations which can be used at low levels in a wide variety of products.
• the food ingredient may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration.
• the enzymes described herein may be used as a food or feed ingredient or in the preparation or production.
• the enzymes may be - or may not be added to - food supplements.
• Feed compositions for monogastric animals typically include compositions comprising plant products which contain phytate.
• compositions include, but are not limited to, cornmeal, soybean meal, rapeseed meal, sunflower meal, cottonseed meal, maize, wheat, barley and sorghum-based feeds.
• the term “pelleting” refers to the production of pellets which can be solid, rounded, spherical and cylindrical tablets, particularly feed pellets and solid, extruded animal feed.
• a known feed pelleting manufacturing process generally includes admixing together food or feed ingredients at least 1 minutes at room temperature, transferring the admixture to a surge bin, conveying the admixture to a steam conditioner (i.e., conditioning), Attorney Docket No.
• NB42148-WO-PCT optionally transferring the steam conditioned admixture to an expander, transferring the admixture to the pellet mill or extruder, and finally transferring the pellets into a pellet cooler.
• pellet refers to a composition of animal feed (usually derived from grain) that has been subjected to a heat treatment, such as a steam treatment (i.e., conditioning), and pressed or extruded through a machine.
• the pellet may incorporate enzyme in the form of a liquid preparation or a dry preparation.
• the dry preparation may be coated or not coated and may be in the form of a granule.
• granule is used for particles composed of enzymes (such as a phytase, for example, any of the engineered phytase polypeptides disclosed herein) and other chemicals such as salts and sugars and may be formed using any of a variety of techniques, including fluid bed granulation approaches to form layered granules.
• specific activity as used herein is the number of enzyme units per ml divided by the concentration of (total) protein in mg/ml. Specific activity values are therefore usually quoted as units/mg.
• isolated means a substance in a form or environment that does not occur in nature and does not reflect the extent to which an isolate has been purified but indicates isolation or separation from a native form or native environment.
• Non-limiting examples of isolated substances include (1 ) any non-naturally occurring substance, (2) any substance including, but not limited to, any host cell, enzyme, engineered enzyme, 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.
• isolated nucleic acid molecule “isolated polynucleotide”, and “isolated nucleic acid fragment” will be used interchangeably and refer to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases.
• An isolated nucleic acid molecule in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
• Attorney Docket No. NB42148-WO-PCT The terms “purify,” “purified,” and purification mean to make substantially pure or clear from unwanted components, material defilement, admixture or imperfection.
• purification generally denotes a nucleic acid or polypeptide that is essentially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation).
• a nucleic acid or polypeptide that gives rise to essentially one band in an electrophoretic gel is “purified.”
• a purified nucleic acid or polypeptide is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight on a molar basis).
• a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique.
• enriched refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.
• peptides “proteins” and “polypeptides are used interchangeably herein and refer to a polymer of amino acids joined together by peptide bonds.
• a “protein” or “polypeptide” comprises a polymeric sequence of amino acid residues.
• the single and 3-letter code for amino acids as defined in conformity with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) is used throughout this disclosure.
• the single letter X refers to any of the twenty amino acids.
• a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code. Mutations can be named by the one letter code for the parent amino acid, followed by a position number and then the one letter code for the variant amino acid. For example, mutating glycine (G) at position 87 to serine (S) is represented as “G087S” or “G87S”.
• a position followed by amino acids listed in parentheses indicates a list of substitutions at that position by any of the listed amino acids.
• 6(L, I) means position 6 can be substituted with a leucine or isoleucine.
• a slash (/) is used to define substitutions, e.g. F/V, indicates that the position may have a phenylalanine or valine at that position.
• corresponding to or “corresponds to” or “correspond to” or “corresponds” refers to an amino acid residue at the enumerated position in a protein or peptide, or an amino acid residue that is analogous, homologous, or equivalent to an enumerated residue in a protein or peptide.
• corresponding region generally refers to an analogous position in a related protein or a reference protein.
• the terms “derived from” and “obtained from” refer to not only a protein produced or producible by a strain of the organism in question, but also a protein encoded by a DNA sequence isolated from such strain and produced in a host organism containing such DNA sequence.
• amino acid refers to the basic chemical structural unit of a protein or polypeptide. The following abbreviations used herein to identify specific amino acids can be found in Table A. Table A.
• transformation refers to the transfer or introduction of a nucleic acid molecule into a host organism.
• the nucleic acid molecule may be introduced as a linear or circular form of DNA.
• the nucleic acid molecule may be a plasmid that replicates autonomously, or it may integrate into the genome of a production host.
• Production hosts containing the transformed nucleic acid are referred to as “transformed” or “recombinant” or “transgenic” organisms or “transformants”.
• the terms “recombinant” and “engineered” refer to an artificial combination of two otherwise separated segments of nucleic acid sequences, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. For example, DNA in which one or more segments or genes have been inserted, either naturally or by laboratory manipulation, from a different molecule, from another part of the same molecule, or an artificial sequence, resulting in the introduction of a new sequence in a gene and subsequently in an organism.
• NB42148-WO-PCT engineered”, “genetically engineered” and “modified for exogenous gene expression” are used interchangeably herein.
• the terms “recombinant construct”, “expression construct”, “recombinant expression construct” and “expression cassette” are used interchangeably herein.
• a recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that are not all found together in nature.
• a construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different than that found in nature.
• Such a construct may be used by itself or may be used in conjunction with a vector.
• a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art.
• a plasmid vector can be used.
• the skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells.
• the skilled artisan will also recognize that different independent transformation events may result in different levels and patterns of expression (Jones et al., (1985) EMBO J 4:2411- 2418; De Almeida et al., (1989) Mol Gen Genetics 218:78-86), and thus that multiple events are typically screened to obtain lines displaying the desired expression level and pattern.
• production host refers to any plant, organism, or cell of any plant or organism, whether human or non-human into which a recombinant construct can be stably or transiently introduced to express a gene. This term encompasses any progeny of a parent cell, which is not identical to the parent cell due to mutations that occur during propagation.
• identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.
• identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the number of matching nucleotides or amino acids between strings of such sequences.
• Identity and similarity can be readily calculated by Attorney Docket No. NB42148-WO-PCT known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D.
• Useful algorithms include the BLAST algorithms (See, Altschul et al., J Mol Biol, 215:403-410, 1990; and Karlin and Altschul, Proc Natl Acad Sci USA, 90:5873-5787, 1993).
• the BLAST program uses several search parameters, most of which are set to the default values.
• the NCBI BLAST algorithm finds the most relevant sequences in terms of biological similarity but is not recommended for query sequences of less than 20 residues (Altschul et al., Nucleic Acids Res, 25:3389-3402, 1997; and Schaffer et al., Nucleic Acids Res, 29:2994-3005, 2001).
• a percent (%) amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the “reference” sequence.
• BLAST algorithms refer to the “reference” sequence as the “query” sequence.
• homologous proteins or “homologous phytases” refers to proteins that have distinct similarity in primary, secondary, and/or tertiary structure. Protein homology can refer to the similarity in linear amino acid sequence when proteins are aligned. Homologous search of protein sequences can be done using BLASTP and PSI-BLAST from NCBI BLAST with threshold (E-value cut-off) at 0.001. (Altschul SF, Madde TL, Shaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI BLAST a new generation of protein database Attorney Docket No. NB42148-WO-PCT search programs.
• CLUSTALW CLUSTALW
• a fast or slow alignment is used with the default settings where a slow alignment.
• multiple sequence alignment may be derived using MAFFT alignment from Geneious® version 10.2.4 with default settings, scoring matrix BLOSUM62, gap open penalty 1.53 and offset value 0.123.
• the MUSCLE program Robot C. Edgar. MUSCLE: multiple sequence alignment with high accuracy and high throughput Nucl. Acids Res.
• engineered phytase polypeptide means that the polypeptide is not naturally occurring and has phytase activity. It is noted that a fragment of the engineered phytase polypeptide is a portion or subsequence of the engineered phytase polypeptide that is capable of functioning like the engineered phytase polypeptide, i.e., it retains phytase activity.
• vector refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types.
• Vectors include, but are not limited to, cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.
• Attorney Docket No. NB42148-WO-PCT An “expression vector” as used herein means a DNA construct comprising a DNA sequence which is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host.
• control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.
• expression refers to the production of a functional end- product (e.g., an mRNA or a protein) in either precursor or mature form. Expression may also refer to translation of mRNA into a polypeptide. Expression of a gene involves transcription of the gene and translation of the mRNA into a precursor or mature protein.
• “Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any signal sequence, pre- or propeptides present in the primary translation product have been removed.
• Precursor refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present.
• Pre- and propeptides may be but are not limited to intracellular localization signals.
• Stable transformation refers to the transfer of a nucleic acid fragment into a genome of a host organism, including both nuclear and organellar genomes, resulting in genetically stable inheritance.
• transient transformation refers to the transfer of a nucleic acid fragment into the nucleus, or DNA- containing organelle, of a host organism resulting in gene expression without integration or stable inheritance.
• a recombinant construct comprising a regulatory sequence functional in a production host operably linked to a nucleotide sequence encoding an engineered phytase polypeptide and fragments thereof as described herein.
• This recombinant construct may comprise a regulatory sequence functional in a production host operably linked to a nucleotide sequence encoding any of the engineered phytase polypeptide and fragments thereof described herein.
• the production host is selected from the group consisting of bacteria, fungi, yeast, plants or algae.
• the production host is the filamentous fungus, Trichoderma reesei.
• promoters Possible initiation control regions or promoters that can be included in the expression vector are numerous and familiar to those skilled in the art.
• a "constitutive promoter” is a promoter that is active under most environmental and developmental conditions.
• An “inducible” or “repressible” promoter is a promoter that is active under environmental or developmental regulation. In some embodiments, promoters are inducible or repressible due to changes in environmental factors including but not limited to, carbon, nitrogen or other nutrient availability, temperature, pH, osmolarity, the presence of heavy metal(s), the concentration of inhibitor(s), stress, or a combination of the foregoing, as is known in the art.
• the inducible or repressible promoters are inducible or repressible by metabolic factors, such as the level of certain carbon sources, the level of certain energy sources, the level of certain catabolites, or a combination of the foregoing as is known in the art.
• the promoter is one that is native to the host cell.
• the promoter can be a native T. reesei promoter such as the cbh1 promoter which is deposited in GenBank under Accession Number D86235.
• promoters useful for fungal expression include, cbh2, egl1, egl2, egl3, egl4, egl5, xyn1, and xyn2, repressible acid phosphatase gene (phoA) promoter of P. chrysogenus (see e.g., Graessle et al., (1997) Appl. Environ.
• phoA repressible acid phosphatase gene
• Microbiol., 63 :753- 756) glucose repressible PCK1 promoter (see e.g., Leuker et al., (1997), Gene, 192:235-240), maltose inducible, glucose-repressible MET3 promoter (see Liu et al., (2006), Eukary. Cell, 5:638-649), pKi promoter and cpc1 promoter.
• Other examples of useful promoters include promoters from A. awamori and A. niger glucoamylase genes (see e.g., Nunberg et al., (1984) Mol. Cell Biol.
• the promoters of the T. reesei xln1 gene may be useful (see e.g., EPA 137280Al).
• DNA fragments which control transcriptional termination may also be derived from various genes native to a preferred production host cell. In certain embodiments, the inclusion of a termination control region is optional. In certain embodiments, the expression vector includes a termination control region derived from the preferred host cell.
• production host means a suitable host for an expression vector or DNA construct comprising a polynucleotide encoding phytase polypeptide or fragment thereof.
• the choice of a production host can be selected from the group consisting of bacteria, fungi, yeast, plants and Attorney Docket No. NB42148-WO-PCT algae. Typically, the choice will depend upon the gene encoding the engineered phytase polypeptide or fragment thereof and its source.
• host strains are preferably filamentous fungal cells.
• “host cell” means both the cells and protoplasts created from the cells of a filamentous fungal strain and particularly a Trichoderma sp. or an Aspergillus sp.
• filamentous fungi refers to all filamentous forms of the subdivision Eumycotina (See, Alexopoulos, C. J. (1962), INTRODUCTORY MYCOLOGY, Wiley, New York). These fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose, and other complex polysaccharides.
• the filamentous fungi of the present invention are morphologically, physiologically, and genetically distinct from yeasts.
• the filamentous fungal parent cell may be a cell of a species of, but not limited to, Trichoderma, (e.g., Trichoderma reesei (previously classified as T. longibrachiatum and currently also known as Hypocrea jecorina), Trichoderma viride, Trichoderma koningii, Trichoderma harzianum); Penicillium sp., Humicola sp. (e.g., Humicola insolens and Humicola grisea); Chrysosporium sp. (e.g., C.
• Trichoderma e.g., Trichoderma reesei (previously classified as T. longibrachiatum and currently also known as Hypocrea jecorina), Trichoderma viride, Trichoderma koningii, Trichoderma harzianum
• Penicillium sp. Humicola sp. (e.g., Humicola insolens and Humicola grise
• Trichoderma or “Trichoderma sp.” refer to any fungal genus previously or currently classified as Trichoderma.
• An expression cassette can be included in the production host, particularly in the cells of microbial production hosts.
• the production host cells can be microbial hosts found within the fungal families and which grow over a wide range of temperature, pH values, and solvent tolerances.
• any of bacteria, yeast, plants, algae, or fungi such as filamentous fungi, may suitably host the expression vector.
• Inclusion of the expression cassette in the production host cell may be used to express the protein of interest so that it may reside intracellularly, extracellularly, or a combination of both inside and outside the cell. Extracellular expression renders recovery of the desired protein from a fermentation product more facile than methods for recovery of protein produced by intracellular expression.
• NB42148-WO-PCT Methods for transforming nucleic acids into filamentous fungi such as Aspergillus spp., e.g., A. oryzae or A. niger, H. grisea, H. insolens, and T. reesei. are well known in the art.
• a suitable procedure for transformation of Aspergillus host cells is described, for example, in EP238023.
• a suitable procedure for transformation of Trichoderma host cells is described, for example, in Steiger et al 2011, Appl. Environ. Microbiol. 77:114-121. Uptake of DNA into the host Trichoderma sp. strain is dependent upon the calcium ion concentration.
• a buffering system such as TE buffer (10 Mm Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0 buffer (morpholinepropanesulfonic acid) and polyethylene glycol (PEG). It is believed that the polyethylene glycol acts to fuse the cell membranes, thus permitting the contents of the medium to be delivered into the cytoplasm of the Trichoderma sp. strain and the plasmid DNA is transferred to the nucleus. This fusion frequently leaves multiple copies of the plasmid DNA integrated into the host chromosome.
• TE buffer 10 Mm Tris, pH 7.4; 1 mM EDTA
• MOPS pH 6.0 buffer (morpholinepropanesulfonic acid)
• PEG polyethylene glycol
• a suspension containing the Trichoderma sp. protoplasts or cells that have been subjected to a permeability treatment at a density of 10 5 to 10 7 /mL, preferably 2 ⁇ 10 6 /mL are used in transformation.
• a volume of 100 ⁇ L of these protoplasts or cells in an appropriate solution e.g., 1.2 M sorbitol; 50 mM CaCl2
• an appropriate solution e.g., 1.2 M sorbitol; 50 mM CaCl2
• PEG a high concentration of PEG is added to the uptake solution.
• From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension. However, it is preferable to add about 0.25 volumes to the protoplast suspension.
• Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride and the like may also be added to the uptake solution and aid in transformation. Similar procedures are available for other fungal host cells. (see, e.g., U.S. Pat. Nos. 6,022,725 and 6,268,328, both of which are incorporated by reference).
• genetically stable transformants are constructed with vector systems whereby the nucleic acid encoding the phytase polypeptide or fragment thereof is stably integrated into a host strain chromosome. Transformants are then purified by known techniques. After the expression vector is introduced into the cells, the transfected or transformed cells are cultured under conditions favoring expression of genes under control of the promoter sequences.
• Culture-conditions are also standard, (e.g., cultures are incubated at approximately 28° C. in appropriate medium in shake cultures or fermenters until desired levels of phytase expression are achieved).
• Preferred culture conditions for a given filamentous fungus are known in the art and may be found in the scientific literature and/or from the source of the fungi such as the American Type Culture Collection and Fungal Genetics Stock Center. After fungal growth has been established, the cells are exposed to conditions effective to cause or permit the expression of a phytase and particularly a phytase as defined herein.
• the inducing agent e.g., a sugar, metal salt or antimicrobial
• the inducing agent is added to the medium at a concentration effective to induce phytase expression.
• An engineered phytase polypeptide or fragment thereof secreted from the host cells can be used, with minimal post-production processing, as a whole broth preparation.
• the preparation of a spent whole fermentation broth of a recombinant microorganism can be achieved using any cultivation method known in the art resulting in the expression of an engineered phytase polypeptide or fragment thereof.
• spent whole fermentation broth is defined herein as unfractionated contents of fermentation material that includes culture medium, extracellular proteins (e.g., enzymes), and cellular biomass. It is understood that the term “spent whole fermentation broth” also encompasses cellular biomass that has been lysed or permeabilized using methods well known in the art.
• a fermentation broth is obtained, the microbial cells and various suspended solids, including residual raw fermentation materials, are removed by conventional separation techniques in order to obtain a phytase solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultra- filtration, extraction, or chromatography, or the like, are generally used.
• an engineered phytase polypeptide or fragment thereof containing culture supernatant is obtained by using any of the methods known to those skilled in the art. Examples of these techniques include, but are not limited to, affinity chromatography (Tilbeurgh et a., (1984) FEBS Lett. 16:215), ion-exchange chromatographic methods (Goyal et al., (1991) Biores. Technol. 36:37; Fliess et al., (1983) Eur. J. Appl. Microbiol. Biotechnol.
• the enzyme containing solution is concentrated using conventional concentration techniques until the desired enzyme level is obtained. Concentration of the enzyme containing solution may be achieved by any of the techniques discussed herein. Exemplary methods of enrichment and purification include but are not limited to rotary vacuum filtration and/or ultrafiltration.
• concentration of the desired protein product may be performed using, e.g., a precipitation agent, such as a metal halide precipitation agent.
• the metal halide precipitation agent, sodium chloride can also be used as a preservative.
• the metal halide precipitation agent is used in an amount effective to precipitate the engineered phytase polypeptide or fragment thereof.
• Exemplary organic compound precipitating agents include: 4-hydroxybenzoic acid, alkali metal salts of 4-hydroxybenzoic acid, alkyl esters of 4-hydroxybenzoic acid, and blends of two or more of these organic compounds.
• the addition of the organic compound precipitation agents can take place prior to, simultaneously with or subsequent to the addition of the metal halide precipitation agent, and the addition of both precipitation agents, organic compound and metal halide, may be carried out sequentially or simultaneously.
• the organic precipitation agents are selected from the group consisting of alkali metal salts of 4-hydroxybenzoic acid, such as sodium or potassium salts, and linear or branched alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 12 carbon atoms, and blends of two or more of these organic compounds.
• Additional organic compounds also include but are not limited to 4- hydroxybenzoic acid methyl ester (named methyl PARABEN), 4-hydroxybenzoic acid propyl ester (named propyl PARABEN).
• methyl PARABEN 4-hydroxybenzoic acid methyl ester
• propyl PARABEN 4-hydroxybenzoic acid propyl ester
• Addition of the organic compound precipitation agent provides the advantage of high flexibility of the precipitation conditions with respect to pH, temperature, concentration, precipitation agent, protein concentration, and time of incubation. Generally, at least about 0.01% w/v and no more than about 0.3% w/v of organic compound precipitation agent is added to the concentrated enzyme solution. After the incubation period, the enriched or purified enzyme is then separated from the dissociated pigment and other impurities and collected by conventional separation techniques, such as filtration, centrifugation, microfiltration, rotary vacuum filtration, ultrafiltration, press filtration, cross membrane microfiltration, cross flow membrane microfiltration, or the like. Further enrichment or purification of the enzyme precipitate can be obtained by washing the precipitate with water.
• the enriched or purified enzyme precipitate is washed with water containing the metal halide precipitation agent, or with water containing the metal halide and the organic compound precipitation agents.
• known methods may be used (e.g. methods disclosed in U.S. Attorney Docket No. NB42148-WO-PCT Pat. Nos. 5,246,853, U.S. Pat. No. 5,475,101 and WO92/06209).
• Gene inactivation may be accomplished by complete or partial deletion, by insertional inactivation or by any other means which renders a gene nonfunctional for its intended purpose (such that the gene is prevented from expression of a functional protein).
• Any gene from a Trichoderma sp. or other filamentous fungal host, which has been cloned can be deleted, for example cbh1, cbh2, egl1 and egl2 genes.
• gene deletion may be accomplished by inserting a form of the desired gene to be inactivated into a plasmid by methods known in the art.
• deletion plasmid is then cut at an appropriate restriction enzyme site(s), internal to the desired gene coding region, and the gene coding sequence or part thereof is replaced with a selectable marker. Flanking DNA sequences from the locus of the gene to be deleted (preferably between about 0.5 to 2.0 kb) remain on either side of the marker gene.
• An appropriate deletion plasmid will generally have unique restriction enzyme sites present therein to enable the fragment containing the deleted gene, including the flanking DNA sequences and the selectable markers gene to be removed as a single linear piece. Depending upon the host cell used post-transcriptional and/or post-translational modifications may be made.
• post-transcriptional and/or post- translational modification is “clipping” or “truncation” of a polypeptide.
• this clipping may result in taking a mature phytase polypeptide and further removing N or C- terminal amino acids to generate truncated forms of the phytase that retain enzymatic activity.
• post-transcriptional or post-translational modifications include, but are not limited to, myristoylation, glycosylation, truncation, lipidation and tyrosine, serine or threonine phosphorylation.
• glycation can affect the activity of phytase when subjected to incubation with glucose or other reducing sugars especially at temperatures above 30oC and neutral or alkaline pH. Protein engineering to eliminate Lysine residues can be used to prevent such modification. An example of this can be found in US 8,507,240. For example, yeast expression can result in highly glycosylated polypeptides resulting in an apparent increased molecular weight.
• the phrase “highly glycosylated” can refer to extensive glycolytic branching (such as, the size and number of glycolytic moieties associated with a particular N-linked glycosylation site) at all or substantially all N-linked glycosylation sites.
• the engineered phytase polypeptide is glycosylated at all or substantially all consensus N-linked glycosylation sites (i.e. an NXS/T consensus N-linked glycosylation site).
• the term “glycan” as used herein refers to a polysaccharide or oligosaccharide, or the carbohydrate section of a glycoconjugate such as a glycoprotein.
• Glycans may be homo- or heteropolymers of monosaccharide residues. They may be linear or branched molecules. A phytase may have varying degrees of glycosylation. It is known that such glycosylations may improve stability during storage and in applications. Extensive The activity of any of the engineered phytase polypeptides or fragments thereof disclosed herein can be determined as discussed above. It is believed that applying a robust engineered phytase polypeptide or fragment thereof to feed in a liquid form is beneficial as compared to applying such a phytase as a coated granule. This coated granule is the current commercial approach to make phytase products suitable for high temperature conditioning and pelleting.
• Benefits of liquid application of robust enzyme include; 1) the enzyme will start to work immediately after ingestion by an animal since it does not have to be released from the coated granule before it can interact with the feed, 2) there is improved distribution of the enzyme throughout the feed, thus, ensuring a more consistent delivery of the enzyme to the animal which is particularly important for young animals that eat small amounts of feed, 3) even distribution in the feed makes it easier to measure the enzyme in the feed, and 4) in the case of a robust phytase, such as the engineered phytase polypeptide and fragment disclosed herein, it may start to degrade phytate already present in the feed. Attorney Docket No.
• the novel engineered phytase polypeptides and fragments thereof are so robust that no special coating or formulation is believed to be needed to apply them to feed prior to conditioning and pelleting since they have been engineered to withstand the stress of conditioning and pelleting used in industrial feed production. Accordingly, the robustness of the novel engineered phytase polypeptides and fragments thereof described herein is such that they can be applied as an uncoated granule or particle or uncoated and unprotected when put into a liquid. It should be noted that the engineered phytase polypeptides and fragments thereof can be formulated inexpensively on a solid carrier without specific need for protective coatings and still maintain activity throughout the conditioning and pelleting process.
• a protective coating to provide additional thermostability when applied in a solid form can be beneficial for obtaining pelleting stability when required in certain regions where harsher conditions are used or if conditions warrant it, e.g., as in the case of super conditioning feed above 90°C.
• the disclosed engineered phytase polypeptides or fragments thereof were derived using a combination of methods and techniques know in the field of protein engineering which include, phylogenetic analysis, site evaluation libraries, combinatorial libraries, high throughput screening and statistical analysis.
• the disclosure relates to an engineered phytase polypeptide or fragment thereof also that has at least 82% sequence identity with the amino acid sequence of SEQ ID NO:1.
• sequence identity also includes 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
• at least 79 % sequence identity also includes 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
• an engineered phytase polypeptide or fragment thereof also that has at least 81% (such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Attorney Docket No. NB42148-WO-PCT 97%, 98%, 99% or 100%) sequence identity with the amino acid sequence of SEQ ID NOs:2, 3, 8, 10, 12, 18, 19, 24, 26, 27, 28, 30, 31, 32, 33, and/or 36.
• an engineered phytase polypeptide or fragment thereof also that has at least 82% (such as 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with the amino acid sequence of SEQ ID NOs:1, 4, 5, 7, 9, 11, 14, 15, 17, 21, 25, 34, and/or 35; c) an engineered phytase polypeptide or fragment thereof also that has at least 83% (such as, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with the amino acid sequence of SEQ ID NO:13; d) an engineered phytase polypeptide or fragment thereof also that has at least 79% (such as, 79%, 80%, 81%, 82%, 83%,
• the polypeptide comprises a core domain of an engineered phytase polypeptide or is a core domain fragment of an engineered phytase polypeptide.
• a “core domain fragment” is herein defined as a polypeptide having one or more amino acids deleted from the amino and/or carboxyl terminus of the polypeptide.
• the phrase “core domain” refers to a polypeptide region encompassing amino acids necessary to maintain the structure and function (such as, phytic acid hydrolysis) of the polypeptide. Amino acids in the core domain can be further modified to improve thermostability or catalytic activity under various conditions such as, without limitation, pH.
• the core domain of the engineered phytase polypeptides or fragment thereof disclosed herein corresponds to amino acid positions 14-325 of SEQ ID NO:1.
• the core domain corresponds to amino acid positions 13-326, 12-327, 11-328, 10-329, 9-330, 8-331, 7-332, 6- 333, 5-334, 4-335, 3-336, 2-337, or 1-338 of SEQ ID NO:1.
• the N- terminus of the core domain corresponds to amino acid position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 of SEQ ID NO:1 and the C-terminus of Attorney Docket No.
• the core domain corresponds to amino acid position 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403,
• an engineered phytase polypeptide or core domain fragment thereof that has at least 78% (such as, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to amino acids 14-325 of SEQ ID NO:6, wherein said amino acid positions correspond to those of SEQ ID NO:1; g) an engineered phytase polypeptide or core domain fragment thereof that has at least 79% (such as, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to amino acids 14-325 of SEQ ID NOs:2, 8, 27, and/or 37,
• NB42148-WO-PCT k an engineered phytase polypeptide or core domain fragment thereof that has at least 84% (such as, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to amino acids 14-325 of SEQ ID NO:29, wherein said amino acid positions correspond to those of SEQ ID NO:1.
• any of the engineered polypeptides or fragments thereof disclosed herein comprise a specific activity of at least about 100 U/mg at pH 3.5.
• the specific activity range (U/mg at pH 3.5) includes, but is not limited to, about 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 2000, etc.
• some of the engineered polypeptides or fragments thereof disclosed herein comprise a specific activity of at least about 100 U/mg at pH 5.5.
• the specific activity range (U/mg at pH 5.5) includes, but is not limited to, about 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 2000, etc.
• any of the engineered phytase polypeptides or fragments thereof disclosed herein may be stable in a liquid form at a pH about 3.0 or lower.
• non-inorganic phosphate-containing diet comprising any of the engineered phytase polypeptides or fragments thereof described herein.
• feed additive enzymes e.g. a phytase is subjected to very harsh conditions as it passes through the digestive track of an animal, i.e. low pH and presence of digestive enzymes.
• Pepsin is one of the most important proteolytic digestive enzymes present in the gastrointestinal tract of monogastric animals.
• Pepsin has low specificity and high pH tolerance in the acidic area (pH 1.5-6.0 stabile up to pH 8.0).
• the engineered phytase polypeptides or fragments thereof described herein are largely resistant against pepsin, which is necessary for good in-vivo performance.
• the non-inorganic phosphate-containing diets comprising any of the engineered phytase polypeptides or fragments thereof described herein may be used (i) alone or (ii) with at least one other enzyme or (iii) further comprising at least one other feed additive component and, optionally, the engineered phytase polypeptide or fragment thereof is present in an amount of at Attorney Docket No.
• NB42148-WO-PCT least 0.1g/ton feed (such as at least about 0.1 g/ton, 0.2 g/ton, 0.3 g/ton, 0.4 g/ton, 0.5 g/ton, 0.6 g/ton, 0.7 g/ton, 0.8 g/ton, 0.9 g/ton, 1 g/ton, 1.1 g/ton, 1.2 g/ton, 1.3 g/ton, 1.4 g/ton, 1.5 g/ton, 1.6 g/ton, 1.7 g/ton, 1.8 g/ton, 1.9 g/ton, 2 g/ton, 2.1 g/ton, 2.2 g/ton, 2.3 g/ton, 2.4 g/ton, 2.5 g/ton, 2.6 g/ton, 2.7 g/ton, 2.8 g/ton, 2.9 g/ton, 3 g
• the phytase is present in the diet in range of about 500 FTU/kg to about 7000 FTU/kg feed or about 1000 FTU/kg feed to about 5000 FTU/kg feed.
• the phytase is present in the feedstuff at more than about 200 FTU/kg feed, suitably more than about 300 FTU/kg feed, suitably more than about 400 FTU/kg feed, suitably more than about 500 FTU/kg feed, suitably more than about 600 FTU/kg feed, suitably more than about 700 FTU/kg feed, suitably more than about 800 FTU/kg feed, suitably more than about 900 FTU/kg feed, suitably more than about 400 FTU/kg feed, suitably more than about 1000 FTU/kg feed, suitably more than about 1100 FTU/kg feed, suitably more than about 1200 FTU/kg feed, suitably more than about 1300 FTU/kg feed, suitably more than about 1400 FTU/kg feed, suitably more than about 1500 FTU
• NB42148-WO-PCT FTU/kg feed suitably more than about 5100 FTU/kg feed, suitably more than about 5200 FTU/kg feed, suitably more than about 5300 FTU/kg feed, suitably more than about 5400 FTU/kg feed, suitably more than about 5500 FTU/kg feed, suitably more than about 5600 FTU/kg feed, suitably more than about 5700 FTU/kg feed.
• “1 FTU” (phytase unit) is defined as the amount of enzyme required to release 1 ⁇ mol of inorganic orthophosphate from a substrate in one minute under the reaction conditions defined in the ISO 2009 phytase assay—A standard assay for determining phytase activity and 1 FTU can be found at International Standard ISO/DIS 30024: 1-17, 2009.
• the enzyme is classified using the E.C. classification above, and the E.C. classification designates an enzyme having that activity when tested in the assay taught herein for determining 1 FTU.
• feed additive “feed additive components”, and/or “feed additive ingredients” are used interchangeably herein.
• Feed additives can be described as products used in animal nutrition for purposes of improving the quality of feed and the quality of food from animal origin, or to improve the animals’ performance and health, e.g. providing enhanced digestibility of the feed materials. Feed additives fall into a number of categories such as sensory additives which stimulate an animal’s appetite so that they naturally want to eat more. Nutritional additives provide a particular nutrient that may be deficient in an animal’s diet. Zootechnical additives improve the overall nutritional value of an animal’s diet through additives in the feed. As used herein, a “non-inorganic phosphate-containing diet” refers to a diet that contains no to substantially no (such as reduced) exogenously added inorganic phosphate, for example, as a feed additive.
• the total P content in the diet may be reduced by about 10-15% (such as any of about 10%, 11%, 12%, 13%, 14%, or 15%) compared to diets that are supplemented with exogenously added inorganic phosphate.
• phosphorus deficient diet or a “diet having low phosphorous content” refers to a diet containing lower levels of the mineral than required for optimal growth. If the diet lacks phosphorus that would lead a phosphorus deficiency in the body of animal, calcium will also not be retained by the animal. Excess Ca can lead to poor phosphorus (P) digestibility and contribute to the formation of insoluble mineral-phytate complexes.
• Feed additive compositions or formulations may also comprise at least one component selected from the group consisting of a protein, a peptide, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride, potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, myo-inositol, magnesium formate, magnesium sorbate, sodium metabisulfite, methyl paraben and propyl paraben.
• At least one other enzyme i.e.
• a xylanase in addition to any of the engineered phytase polypeptides or fragments thereof disclosed herein
• the feed additive compositions or formulations disclosed herein can include, but are not limited to, a xylanase, amylase, another phytase, beta-glucanase, and/or a protease.
• Xylanase is the name given to a class of enzymes that degrade the linear polysaccharide ⁇ -1,4-xylan into xylose, thus breaking down hemicellulose, one of the major components of plant cell walls.
• Xylanases e.g., endo- ⁇ -xylanases (EC 3.2.1.8) hydrolyze the xylan backbone chain.
• the xylanase may be any commercially available xylanase.
• the xylanase may be an endo-1,4-P-d-xylanase (classified as E.G. 3.2.1.8) or a 1,4 ⁇ -xylosidase (classified as E.G. 3.2.1.37).
• the disclosure relates to a composition comprising any of the engineered phytase polypeptides or fragments thereof disclosed herein in combination with an endoxylanase, e.g.
• the xylanase may be a xylanase from Bacillus, Trichodermna, Therinomyces, Aspergillus, Humicola and Penicillium.
• the xylanase may be the xylanase in Axtra XAP® or Avizyme 1502®, both commercially available products from Danisco A/S.
• the xylanase may be a mixture of two or more xylanases.
• the xylanase is an endo-1,4- ⁇ -xylanase or a 1,4- ⁇ - xylosidase.
• the disclosure relates to a non-inorganic phosphate-containing diet comprising any of the engineered phytase polypeptides or fragments thereof disclosed herein and a xylanase.
• the non-inorganic phosphate-containing diet comprises 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, and greater than 750 xylanase units/g of composition.
• the non-inorganic phosphate-containing diet comprises 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500-7000, 7000-7500, 7500-8000, and greater than 8000 xylanase units/g composition.
• one xylanase unit is the amount of enzyme that releases 0.5 ⁇ mol of reducing sugar equivalents (as xylose by the Dinitrosalicylic acid (DNS) assay- reducing sugar method) from an oat-spelt-xylan substrate per min at pH 5.3 and 50° C.
• DMS Dinitrosalicylic acid
• Amylase is a class of enzymes capable of hydrolysing starch to shorter-chain oligosaccharides, such as maltose.
• amylase includes ⁇ -amylases (E.C. 3.2.1.1), G4-forming amylases (E.C. 3.2.1.60), ⁇ - amylases (E.C. 3.2.1.2) and ⁇ -amylases (E.C. 3.2.1.3).
• Amylases may be of bacterial or fungal origin, or chemically modified or protein engineered mutants.
• the amylase may be a mixture of two or more amylases.
• the amylase may be an amylase, e.g.
• the ⁇ - amylase may be the ⁇ -amylase in Axtra XAP® or Avizyme 1502®, both commercially available Attorney Docket No. NB42148-WO-PCT products from Danisco A/S.
• the amylase may be a pepsin resistant ⁇ -amylase, such as a pepsin resistant Trichoderma (such as Trichoderma reesei) alpha amylase.
• amylase unit is the amount of enzyme that releases 1 mmol of glucosidic linkages from a water insoluble cross-linked starch polymer substrate per min at pH 6.5 and 37° C. (this may be referred to herein as the assay for determining 1 AU).
• disclosure relates to a non-inorganic phosphate-containing diet comprising any of the engineered phytase polypeptides or fragments thereof disclosed herein and an amylase.
• disclosure relates to a non-inorganic phosphate-containing diet comprising any of the engineered phytase polypeptides or fragments thereof disclosed herein, xylanase and amylase.
• the composition comprises 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, and greater than 750 amylase units/g composition.
• the non-inorganic phosphate-containing diet comprises 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500-7000, 7000-7500, 7500-8000, 8000-8500, 8500-9000, 9000-9500, 9500-10000, 10000-11000, 11000-12000, 12000-13000, 13000-14000, 14000-15000 and greater than 15000 amylase units/g composition.
• protease as used herein is synonymous with peptidase or proteinase.
• the protease may be a subtilisin (E.C.
• the protease is a subtilisin. Suitable proteases include those of animal, vegetable or microbial origin. Chemically modified or protein engineered mutants are also suitable.
• the protease may be a serine protease or a metalloprotease. e.g., an alkaline microbial protease or a trypsin-like protease.
• compositions comprising any of the engineered phytase polypeptides or fragments thereof disclosed herein and one or more protease.
• alkaline proteases are subtilisins, especially those derived from Bacillus sp., e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309 (see, e.g., U.S. Pat. No. 6,287,841), subtilisin 147, and subtilisin 168 (see, e.g., WO 89/06279).
• subtilisins especially those derived from Bacillus sp., e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309 (see, e.g., U.S. Pat. No. 6,287,841), subtilisin 147, and subtilisin 168 (see, e.g., WO 89/06279).
• trypsin-like proteases Attorney Docket No.
• NB42148-WO-PCT are trypsin (e.g., of porcine or bovine origin), and Fusarium proteases (see, e.g., WO 89/06270 and WO 94/25583).
• useful proteases also include but are not limited to the variants described in WO 92/19729 and WO 98/20115.
• the protease is selected from the group consisting of subtilisin, a bacillolysin, an alkine serine protease, a keratinase, and a Nocardiopsis protease.
• one protease unit is the amount of enzyme that liberates from the substrate (0.6% casein solution) one microgram of phenolic compound (expressed as tyrosine equivalents) in one minute at pH 7.5 (40 mM Na2PO4/lactic acid buffer) and 40° C. This may be referred to as the assay for determining 1 PU.
• disclosure relates to a non-inorganic phosphate-containing diet comprising any of the engineered phytase polypeptides or fragments thereof disclosed herein and a protease.
• disclosure relates to a non-inorganic phosphate-containing diet comprising any of the engineered phytase polypeptides or fragments thereof disclosed herein and a xylanase and a protease.
• the disclosure relates to a non- inorganic phosphate-containing diet comprising any of the engineered phytase polypeptides or fragments thereof disclosed herein and an amylase and a protease.
• the disclosure relates to a non-inorganic phosphate-containing diet comprising any of the engineered phytase polypeptides or fragments thereof disclosed herein and a xylanase, an amylase and a protease.
• the non-inorganic phosphate-containing diet comprises about 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, and greater than 750 protease units/g composition.
• the non-inorganic phosphate-containing diet comprises about 500- 1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500- 5000, 5000-5500, 5500-6000, 6000-6500, 6500-7000, 7000-7500, 7500-8000, 8000-8500, 8500- 9000, 9000-9500, 9500-10000, 10000-11000, 11000-12000, 12000-13000, 13000-14000, 14000- 15000 and greater than 15000 protease units/g composition.
• the diet can have reduced (such as substantially reduced) inorganic phosphate levels relative to total phosphorous levels recommended by the National Research Council (NRC) for ruminant animals (such as cattle, for example, dairy cows).
• the diets contain from between 0.2% to about 75% inorganic phosphate Attorney Docket No.
• NB42148-WO-PCT levels relative to those recommended by the National Research Council (NRC) for ruminant animals (such as cattle, for example, dairy cows), such as any of about 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,
• a non-inorganic phosphate-containing diet for use in animal feed comprising at least one polypeptide having phytase activity as described herein, used either alone or in combination with at least one direct fed microbial or in combination with at least one other enzyme or in combination with at least one direct fed microbial and at least one other enzyme, wherein the feed additive composition comprises may be in any form such as a granulated particle.
• Such granulated particles may be produced by a process selected from the group consisting of high shear granulation, drum granulation, extrusion, spheronization, fluidized bed agglomeration, fluidized bed spray coating, spray drying, freeze drying, prilling, spray chilling, spinning disk atomization, coacervation, tableting, or any combination of the above processes.
• particles of the granulated feed additive composition can have a mean diameter of greater than 50 microns and less than 2000 microns
• animal feed may include plant material such as corn, wheat, sorghum, soybean, canola, sunflower or mixtures of any of these plant materials or plant protein sources for poultry, pigs, ruminants, aquaculture and pets.
• an “effective amount” as used herein refers to the amount of an active agent (such as, a phytase, e.g.
• any of the engineered phytase polypeptides disclosed herein required to confer improved performance on an animal on one or more metrics, either alone or in combination with one or more other active agents (such as, without limitation, one or more additional enzyme(s), one or more DFM(s), one or more essential oils, etc.).
• animal performance may be determined by any metric such as, without limitation, the feed efficiency and/or weight gain of the animal and/or by the feed conversion ratio and/or milk production levels and/or by the digestibility of a nutrient in a feed (e.g., amino acid digestibility or phosphorus digestibility) and/or digestible energy or metabolizable energy in a feed and/or by nitrogen retention and/or by animals’ ability to avoid the negative effects of diseases or by the immune response of the subject.
• a nutrient in a feed e.g., amino acid digestibility or phosphorus digestibility
• digestible energy or metabolizable energy in a feed e.g., by nitrogen retention and/or by animals
• a phytase polypeptide or a fragment thereof comprising phytase activity such as a phytase polypeptide or a fragment thereof comprising at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO:1
• phytase activity such as a phytase polypeptide or a fragment thereof comprising at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO:1
• the phrase “improved phosphorous utilization” refers to an increased proportion of consumed phosphorus being retained in an animal’s body and/or less phosphorus being excreted in the animal’s in urine or feces.
• the method results in about a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, or 125%, inclusive of all values falling in between these percentages, improvement in phosphorous utilization relative to phosphate utilization in a lactating ruminant animal
• the phytase polypeptide or fragment thereof is administered in conjunction with a diet having low phosphorous content (such as a diet that has not been supplemented with exogenously added inorganic phosphate).
• Phosphorous utilization can be measured using any means known in the art, including the methods described in the Examples section.
• Attorney Docket No. NB42148-WO-PCT Further provided herein are methods for improving protein utilization in a lactating ruminant animal comprising administering to the animal a phytase polypeptide or a fragment thereof comprising phytase activity (such as a phytase polypeptide or a fragment thereof comprising at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO:1).
• improved protein utilization refers to an increased proportion of consumed protein being retained in the animal’s body or secreted into milk protein and/or less protein (such as nitrogen) being excreted in the animal’s in urine or feces.
• the method results in about a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, or 125%, inclusive of all values falling in between these percentages, improvement in protein utilization relative to protein utilization in a lactating ruminant animal that has not been
• the phytase polypeptide or fragment thereof is administered in conjunction with a diet having low phosphorous content (such as a diet that has not been supplemented with exogenously added inorganic phosphate).
• a diet having low phosphorous content such as a diet that has not been supplemented with exogenously added inorganic phosphate.
• Protein utilization can be measured using any means known in the art, including the methods described in the Examples section.
• methods for improving digestibility of crude protein in a lactating ruminant animal comprising administering to the animal a phytase polypeptide or a fragment thereof comprising phytase activity (such as a phytase polypeptide or a fragment thereof comprising at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO:1).
• the phrase “improved digestibility of crude protein” refers to an increased absorption of protein (such as amino acids) from the protein present in the animal’s diet.
• the method results in about a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 11
• NB42148-WO-PCT of crude protein relative to the digestibility of crude protein in a lactating ruminant animal that has not been administered the phytase polypeptide or a fragment thereof.
• the phytase polypeptide or fragment thereof is administered in conjunction with a diet having low phosphorous content (such as a diet that has not been supplemented with exogenously added inorganic phosphate).
• Digestibility of crude protein can be measured using any means known in the art, including the methods described in the Examples section.
• phytase polypeptide or a fragment thereof comprising phytase activity such as a phytase polypeptide or a fragment thereof comprising at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO:1.
• phytase activity such as a phytase polypeptide or a fragment thereof comprising at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO:1.
• the phrase “decreased excretion of crude protein” means a decrease in the amount of protein excreted in the feces and/or urine of the animal.
• the method results in about a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, inclusive of all values falling in between these percentages, decreased excretion of crude protein relative to the excretion of crude protein in a lactating ruminant animal that has not been administered the phytas
• the phytase polypeptide or fragment thereof is administered in conjunction with a diet having low phosphorous content (such as a diet that has not been supplemented with exogenously added inorganic phosphate).
• Excretion of crude protein can be measured using any means known in the art, including the methods described in the Examples section.
• methods for improving milk protein content in a lactating ruminant animal comprising administering to the animal a phytase polypeptide or a fragment thereof comprising phytase activity (such as a phytase polypeptide or a fragment thereof comprising at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO:1).
• the phrase “improved milk protein content” means the amount of protein content in the ruminant animal’s milk is greater than that of a control group that has not been administered the phytase polypeptide or a fragment thereof.
• the method Attorney Docket No.
• NB42148-WO-PCT results in about a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, or 125%, inclusive of all values falling in between these percentages, improved milk protein content relative to the milk protein content in a lactating ruminant
• the phytase polypeptide or fragment thereof is administered in conjunction with a diet having low phosphorous content (such as a diet that has not been supplemented with exogenously added inorganic phosphate).
• Milk protein content can be measured using any means known in the art, including the methods described in the Examples section.
• methods for improving milk protein yield in a lactating ruminant animal comprising administering to the animal a phytase polypeptide or a fragment thereof comprising phytase activity (such as a phytase polypeptide or a fragment thereof comprising at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO:1).
• the phrase “improved milk protein yield” means total milk protein production per day is higher and which is calculated as the product of milk protein x milk production/day.
• the method results in about a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%,
• the phytase polypeptide or fragment thereof is administered in conjunction with a diet having low phosphorous content (such as a diet that has not been supplemented with exogenously added inorganic phosphate).
• Milk protein yield can be measured using any means known in the art, including the methods described in the Examples section.
• Attorney Docket No. NB42148-WO-PCT Still further provided herein are methods for improving calcium digestibility in a lactating ruminant animal comprising administering to the animal a phytase polypeptide or a fragment thereof comprising phytase activity (such as a phytase polypeptide or a fragment thereof comprising at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO:1).
• the phrase “improved calcium digestibility” means an increased absorption of calcium relative to total calcium intake and corresponding less calcium excretion in the feces.
• the method results in about a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%
• the phytase polypeptide or fragment thereof is administered in conjunction with a diet having low phosphorous content (such as a diet that has not been supplemented with exogenously added inorganic phosphate).
• a diet having low phosphorous content such as a diet that has not been supplemented with exogenously added inorganic phosphate.
• Calcium digestibility can be measured using any means known in the art, including the methods described in the Examples section. Ruminants have a stomach with four chambers, namely the rumen, reticulum, omasum and abomasum. In the first two chambers, the rumen and the reticulum, food is mixed with saliva and separates into layers of solid and liquid material. Solids clump together to form the cud, or bolus.
• the cud is then regurgitated, chewed slowly to completely mix it with saliva, which further breaks down fibers.
• Fiber, especially cellulose is broken down into glucose in these chambers by the enzymes produced by commensal bacteria, protozoa and fungi.
• the broken- down fiber which is now in the liquid part of the contents, then passes through the rumen and reticulum into the next stomach chamber, the omasum, where water is removed.
• the food in the abomasum is digested much like it would be in the human stomach.
• the abomasum has a pH of around 2.0 and therefore possesses an environment capable of denaturing most, if not all, polypeptides.
• the processed food is finally sent to the small intestine, where the absorption of the nutrients occurs.
• a method for degrading phytate in the abomasum of a ruminant animal comprising administering to the animal any of the phytase polypeptides or functional fragments thereof provided herein (such as a phytase polypeptide or a fragment thereof comprising phytase activity (such as a phytase polypeptide or a fragment thereof comprising at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO:1)).
• degrading phytate in the abomasum of a ruminant animal means a substantial amount (for example any of about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of phytate is degraded in the abomasum of the animal compared to the total amount of degraded phytate in the diet and/or compared to the amount of phytate degraded in the rumen.
• the phytase polypeptide or functional fragment thereof can degrade phytase in the abomasum at about double the rate (such as any of about 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 times, or greater, the rate) of a control phytase polypeptide or a fragment thereof that does not comprise at least 82% sequence identity of the amino acid sequence set forth in SEQ ID NO:1.
• the phytase polypeptide or functional fragment thereof can degrade phytase in the abomasum at about double the disappearance (as a percentage, such as any of about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the amount of phytate remaining after passage through the rumen) as compared to a phytase polypeptide or a fragment thereof that does not comprise at least 82% sequence identity of the amino acid Attorney Docket No.
• the phytate is degraded in the presence of pepsin. In other embodiments, the phytate is degraded at a pH of about 1.5 to 3 (such as any of about 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3).
• the improvement in performance parameters may be in respect to a control in which the feed used does not comprise a phytase (such as any of the phytase or functional fragments thereof disclosed herein).
• the terms “mineralization” or “mineralization” encompass mineral deposition or release of minerals.
• Minerals may be deposited or released from the body of the animal. Minerals may be released from the feed. Minerals may include any minerals necessary in an animal diet, and may include calcium, copper, sodium, phosphorus, iron and nitrogen. Nutrient digestibility as used herein 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.
• 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 phosphorus digestibility, starch digestibility, fat digestibility, protein digestibility, and amino acid digestibility.
• Digestible phosphorus (P) can be defined as ileal digestible P which is the proportion of total P intake absorbed at the end of the ileum by an animal or the fecal digestible P which is the proportion of total P intake that is not excreted in the feces.
• survival means the number of subjects remaining alive.
• improved survival is another way of saying “reduced mortality”.
• carcass yield means the amount of carcass as a proportion of the live body weight, after a commercial or experimental process of slaughter.
• carcass means the body of an animal that has been slaughtered for food, with the head, entrails, part of the limbs, and feathers or skin removed.
• meat yield means the amount of edible meat as a proportion of the live body weight, or the amount of a specified meat cut as a proportion of the live body weight.
• An “increased weight gain” refers to an animal having increased body weight on being fed feed comprising a feed additive composition compared with an animal being fed a feed without said feed additive composition being present.
• animal feed composition can comprise one or more feed materials selected from the group comprising a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and/or large grains such as maize or sorghum; b) by products from cereals, such as corn gluten meal, Distillers Dried Grains with Solubles (DDGS) (particularly corn based Distillers Dried Grains with Solubles (cDDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) protein obtained from sources such as soya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats obtained from vegetable and animal sources; and
• a feed can contain or comprise one or more cereal byproducts of a distillation process.
• a “cereal byproduct of a distillation process” refers to a co- product or by-product from a fermentation of a feedstock or biomass (e.g., fermentation of grain or a grain mixture that produces a product alcohol).
• a cereal byproduct of a distillation process may also refer to an animal feed product produced from a process of making a product alcohol (e.g., ethanol, butanol, isobutanol, etc.).
• the phytase or functional fragment thereof (such as any of those disclosed herein) is added to the cereal (such as, without limitation, grains, e.g., corn, wheat, rye, barley, oats, and mixtures thereof) during a saccharification and/or fermentation process.
• saccharification refers to the process of hydrolyzing polysaccharides and/or oligosaccharides, for example, alpha- 1 ,4-glucosidic bonds of glycogen, or starch.
• Enzymes used during saccharification may include, without limitation, enzymes capable of hydrolyzing cellulosic or lignocellulosic materials as well Attorney Docket No.
• NB42148-WO-PCT e.g., amylases, glucoamylases, proteases, etc.
• "Fermentation” as used herein means a carbon source capable of being metabolized by a microorganism (e.g., yeast) for the production of a fermentative alcohol (e.g., ethanol).
• Suitable fermentable carbon sources include, but are not limited to, products of saccharification, for example, monosaccharides such as glucose or fructose; disaccharides such as lactose or sucrose; oligosaccharides; polysaccharides such as starch or cellulose; C5 sugars such as xylose and arabinose; one carbon substrates including methane; and mixtures thereof.
• a premix as referred to herein may be a composition composed of microingredients such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products, and other essential ingredients. Premixes are usually compositions suitable for blending into commercial rations.
• the term "contacted” refers to the indirect or direct application of any of the engineered phytase polypeptides or fragments thereof (or composition comprising any of the engineered phytase polypeptides or fragments thereof) to a product (e.g. the feed).
• Examples of application methods which may be used include, but are not limited to, treating the product in a material comprising the feed additive composition, direct application by mixing the feed additive composition with the product, spraying the feed additive composition onto the product surface or dipping the product into a preparation of the feed additive composition.
• the feed additive composition of the present invention is preferably admixed with the product (e.g. feedstuff).
• the feed additive composition may be included in the emulsion or raw ingredients of a feedstuff.
• it is important that the composition is made available on or to the surface of a product to be affected/treated. This allows the composition to impart a performance benefit.
• any of the engineered phytase polypeptides or fragments thereof described herein may be applied to intersperse, coat and/or impregnate a product (e.g. a diet that contains no or substantially no inorganic phosphorus or feedstuff or raw ingredients of a feedstuff) with a controlled amount of said enzyme.
• a product e.g. a diet that contains no or substantially no inorganic phosphorus or feedstuff or raw ingredients of a feedstuff
• the feed additive composition can be homogenized to produce a powder.
• the powder may be mixed with other components known in the art.
• the powder, or Attorney Docket No. NB42148-WO-PCT mixture comprising the powder may be forced through a die and the resulting strands are cut into suitable pellets of variable length.
• the pelleting step may include a steam treatment, or conditioning stage, prior to formation of the pellets.
• the mixture comprising the powder may be placed in a conditioner, e.g. a mixer with steam injection.
• the mixture is heated in the conditioner up to a specified temperature, such as from 60-100oC, typical temperatures would be 70oC, 80oC, 85oC, 90oC or 95oC.
• the residence time can be variable from seconds to minutes. It will be understood that any of the engineered phytase polypeptides or fragments thereof (or composition comprising any of the engineered phytase polypeptides or fragments thereof) described herein are suitable for addition to any appropriate feed material.
• the granule may be introduced into a feed pelleting process wherein the feed pretreatment process may be conducted between 70°C and 95°C for up to several minutes, such as between 85°C and 95°C.
• any of the engineered phytase polypeptides or fragments thereof can be present in the feed in the range of 1 ppb (parts per billion) to 10 % (w/w) based on pure enzyme protein.
• the engineered phytase polypeptides or fragments thereof are present in the feedstuff is in the range of 1-100 ppm (parts per million).
• a preferred dose can be 1-20 g of an engineered phytase polypeptide or fragment thereof per ton of feed product or feed composition or a final dose of 1 – 20 ppm engineered phytase polypeptide or fragment thereof in the final feed product.
• an engineered phytase polypeptide or fragment thereof is present in the feed should be at least about 50 – 10,000 FTU/kg corresponding to roughly 0.1 to 20 mg engineered phytase polypeptide or fragment thereof protein/kg. Ranges can include, but are not limited to, any combination of the lower and upper ranges discussed above.
• Formulations and/or preparations comprising any of the engineered phytase polypeptides or fragments thereof and compositions described herein may be made in any suitable way to ensure that the formulation comprises active phytase enzymes.
• Such formulations may be as a liquid, a dry powder or a granule which may be uncoated/unprotected or may involve the use of a thermoprotectant coating depending upon the processing conditions.
• the engineered phytase polypeptides and fragments thereof can be formulated inexpensively on a Attorney Docket No. NB42148-WO-PCT solid carrier without specific need for protective coatings and still maintain activity throughout the conditioning and pelleting process.
• a protective coating to provide additional thermostability when applied in a solid form can be beneficial for obtaining pelleting stability when required in certain regions where harsher conditions are used or if conditions warrant it, e.g., as in the case of super conditioning feed above 90°C.
• Feed additive composition described herein can be formulated to a dry powder or granules as described in WO2007/044968 (referred to as TPT granules) or WO1997/016076 or WO1992/012645 (each of which is incorporated herein by reference).
• the feed additive composition may be formulated to a granule for feed compositions comprising: a core; an active agent (for example, a phytase, such as any of the engineered phytase polypeptides disclosed herein); and at least one coating, the active agent of the granule retaining at least 50% activity, at least 60% activity, at least 70% activity, at least 80% activity after conditions selected from one or more of a) a feed pelleting process, b) a steam-heated feed pretreatment process, c) storage, d) storage as an ingredient in an unpelleted mixture, and e) storage as an ingredient in a feed base mix or a feed premix comprising at least one compound selected from trace minerals, organic acids, reducing sugars, vitamins, choline chloride, and compounds which result in an acidic or a basic feed base mix or feed premix.
• an active agent for example, a phytase, such as any of the engineered phytase polypeptides disclosed
• At least one coating may comprise a moisture hydrating material that constitutes at least 55% w/w of the granule; and/or at least one coating may comprise two coatings.
• the two coatings may be a moisture hydrating coating and a moisture barrier coating.
• the moisture hydrating coating may be between 25% and 60% w/w of the granule and the moisture barrier coating may be between 2% and 15% w/w of the granule.
• the moisture hydrating coating may be selected from inorganic salts, sucrose, starch, and maltodextrin and the moisture barrier coating may be selected from polymers, gums, whey and starch.
• the granule may be introduced into a feed pelleting process wherein the feed pretreatment process may be conducted between 70°C and 95°C for up to several minutes, such as between 85°C and 95°C.
• the feed additive composition may be formulated to a granule for animal feed comprising: a core; an active agent, the active agent of the granule retaining at least 80% activity after storage and after a steam-heated pelleting process where the granule is an ingredient; a Attorney Docket No.
• the NB42148-WO-PCT moisture barrier coating and a moisture hydrating coating that is at least 25% w/w of the granule, the granule having a water activity of less than 0.5 prior to the steam-heated pelleting process.
• the granule may have a moisture barrier coating selected from polymers and gums and the moisture hydrating material may be an inorganic salt.
• the moisture hydrating coating may be between 25% and 45% w/w of the granule and the moisture barrier coating may be between 2% and 10% w/w of the granule.
• the composition is in a liquid formulation suitable for consumption preferably such liquid consumption contains one or more of the following: a buffer, salt, sorbitol and/or glycerol.
• the feed additive composition may be formulated by applying, e.g. spraying, the enzyme(s) onto a carrier substrate, such as ground wheat for example.
• the feed additive composition may be formulated as a premix.
• the premix may comprise one or more feed components, such as one or more minerals and/or one or more vitamins.
• a direct fed microbial (“DFM”) and/or an engineered phytase polypeptide or fragment thereof are formulated with at least one physiologically acceptable carrier selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or a wheat component, sucrose, starch, Na2SO4, Talc, PVA, sorbitol, benzoate, sorbate, glycerol, sucrose, propylene glycol, 1,3-propane diol, glucose, parabens, sodium chloride, citrate, acetate, phosphate, calcium, metabisulfite, formate and mixtures thereof.
• physiologically acceptable carrier selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or a wheat component, sucrose, starch, Na2SO4, Talc, PVA, sorbitol, benzoate, sorbate, glycerol, sucrose, propylene glycol, 1,
• Example 1 Materials and Methods This Example describes the materials and methods used to produce the results in Examples 2 and 3. The study was carried out in accordance with the European Directive 2010/63 EU and the Dutch regulations for the care and use of animals in research.
• Cows were kept in a free stall barn equipped with cubicles (1.10 ⁇ 2.5 m) bedded with chopped straw, rubber floors and had free access to water. Cows were monitored for health daily and any signs of clinical disease were recorded and treated appropriately.
• Treatment diets comprised of a control diet (CON) formulated without supplemental Pi and two experimental diets based on the CON diet but supplemented with a commercial phytase (SEQ ID NO:26) at a targeted dose level of 2,000 or 5,000 phytase units (FTU) per kilogram of total ration on a DM basis.
• CON control diet
• SEQ ID NO:26 commercial phytase
• FTU phytase units
• the phytase was a biosynthetic bacterial 6-phytase, PhyG (Danisco Animal Nutrition & Health, IFF Inc., The Netherlands), expressed in Trichoderma reesei.
• Forage and concentrates were fed to the cows separately. Cows had access to individual Calan gates (American Calan, Northwood, NH) to measure the intake of the forage, and to an automated concentrate dispenser system (Hotraco, Hegelsom, The Netherlands) to supply and measure daily concentrate consumption.
• the forage component comprised a mixture of a constant grass silage to corn silage ratio (30:70 on a DM basis) and was offered ad libitum.
• Table 1 The chemical composition of the forages is shown in Table 1. Table 1.
• composition 1 of forages Attorney Docket No. NB42148-WO-PCT Item, g/kg dry matter Grass Corn (unless otherwise stated) silage silage Dry matter g/kg 604 332 Ash 94 42 Crude protein 157 72 Crude fat 37 31 Neutral detergent fibre 511 349 Acid detergent fibre 295 211 Acid detergent lignin 28 23 Starch n.d. 349 Sugar 105 14 Calcium 2 4.4 1.7 Phosphorus (P) 2 3.0 1.8 Phytate-P 3 0.0 0.0 1 Analyzed by Eurofins Agro NL (Wageningen, The Netherlands) and based on near infrared spectroscopy.
• the concentrates were manufactured by ABZ Diervoeding (Leusden, The Netherlands) and pelleted (exit temperature between 70 and 75°C).
• the concentrates were supplied three times daily (at approximately 0500, 1230 and 1830 h) via the automated dispenser system into separate feeding buckets, in amounts that were individualized per cow based on fat- and protein-corrected milk (FPCM)-yield.
• ingredients were sampled for the analysis of PP and total P content.
• concentrates were optimized to contain a minimum of 2.3 g/kg of PP and a maximum of 3.1 g/kg of total P (equal to 2.8 g/kg DM on a total ration basis).
• Concentrates were formulated to contain phytate-rich ingredients with low ruminal degradability such as formaldehyde treated rapeseed meal and hydrothermal pressure-treated sunflower seed meal.
• the external marker titanium dioxide (TiO2) was added to the concentrates, at a level of 7 g/kg (as is).
• the ingredients and chemical composition of the concentrates is given in Table 2, whilst the calculated chemical composition of the total rations is presented in Table 3.
• the total rations were formulated to meet nutrient requirements as Attorney Docket No.
• the total rations were formulated to contain a low P content (without inorganic phosphate in the concentrate) that represented approximately 90% of the total P requirement according to the Dutch guideline (COMV, 2005).
• the total rations contained P at 2.6 g/kg DM in all treatments.
• a second important factor was to ensure that the diets were representative of commercial diets and that they contained rumen by-pass phytate-rich ingredients such as sunflower meal and rapeseed meal.
• the employed phytase dose levels (2,000 and 5,000 FTU/kg DM) were selected based on the dose levels of other exogenous microbial 6-phytases that have been reported in the literature to improve P digestibility in dairy cows (Brask-Pedersen et al., 2013; Winter et al. 2015) and consideration of the economic feasibility of including PhyG phytase in dairy cow diets.
• Experimental design The experiment was carried out as a randomized block design with three dietary treatments and 10 blocks (replicates) per treatment. The experiment comprised an 18-d pre-period for the collection of data to facilitate the allocation of cows to the treatments, Attorney Docket No.
• NB42148-WO-PCT followed by a 19-d experimental period comprising a 14-d diet adaptation phase, as is recommended for digestibility trials (GfE, 1991), and 5 days of feces collection.
• all cows were fed ad libitum with a mixture of grass silage and corn silage in the same proportions as mentioned above and were supplemented with concentrates (as described above but without supplemental phytase) based on FPCM production.
• Milk yield was individually recorded at each milking using calibrated electronic milk meters (DemaTron 70, GEA, Düsseldorf, Germany). Milk samples were collected per week from each cow on Monday evening, Tuesday morning, Wednesday evening and Thursday morning. Milk samples were preserved with a solution containing sodium azide and bronopol (0.3 mL of solution added per 50 mL of milk) and analyzed for fat, protein, lactose, urea, and somatic cell count by an accredited Dutch laboratory for monitoring milk quality (Qlip, Zutphen, The Netherlands), using Fourier transform infrared spectroscopy (MilkoScan FT6000/7, Foss Electric, Hiller ⁇ d, Denmark).
• the concentrates were produced in one batch each, immediately sampled and analyzed.
• the chemical composition of these forages and concentrate samples was used to calculate the chemical composition of the total rations.
• additional samples of the forages and concentrates were collected for the determination of the apparent total tract digestibility (ATTD) of chemical constituents.
• Forages were sampled daily and concentrates every two days. Samples were stored at ⁇ 20°C until later analysis.
• forages were thawed at room temperature, pooled per type of forage by mixing equal amounts on a FM basis, freeze-dried for approximately 96 h in a Attorney Docket No.
• NB42148-WO-PCT Zirbus sublimator 3-4-5/20 Zirbus Technology Benelux B. V., Tiel, Netherlands
• Retsch ZM200 grinder Retsch Benelux, Aartselaar, Belgium
• the forage and concentrate samples were analyzed by Schothorst Feed Research (Lelystad, the Netherlands).
• the DM content was determined by drying at 103°C to constant weight according to method ISO 6496 (ISO, 1998). Crude ash was determined gravimetrically after ashing the samples in a muffler furnace for 3 h at 550°C, according to method ISO 5984 (ISO, 2002).
• the N content was determined by the Dumas method using a macro determinator (LECO CM928 MLC, LECO, Michigan, USA) according to method ISO 16634 (ISO, 2016), and the CP content was calculated as N ⁇ 6.25.
• the starch content (except in grass silage) was determined by the amylo-glucosidase method according to the procedures of Englyst et al. (1992), and sugar content was determined according to the Luff-Schoorl method.
• Crude fat (CFat) was determined by ether extraction after acid hydrolysis, according to method ISO 11085 (ISO, 2015).
• the NDF content was exclusive of residual ash and a heat-stable ⁇ -amylase was added during NDF extraction, according to ISO 16472 (ISO, 2006).
• the ADF content was exclusive of ash and determined according to ISO 13906 (ISO, 2008).
• the P content was determined based on the colorimetric method according to ISO 6491 (ISO, 1998) and contents of Ca and TiO2 were determined based on atomic absorption spectroscopy according to ISO 6869 (ISO, 2000).
• the content of PP in forages and concentrates was analyzed at Danisco Animal Nutrition Research Centre (Brabrand, Denmark) using the HPLC method described by Christensen et al. (2020) modified from Skoglund et al. (1998).
• Fecal sampling commenced at 0900 and 1300 h on d 1, 3 and 5; and at 1100 and 1430 h on d 2 and 4. This sampling pattern was applied to account for diurnal and day-to-day variations in marker excretion (Glindemann et al., 2009). Samples were immediately frozen at -20°C and stored until later analysis. At the end of the experiment, fecal Attorney Docket No. NB42148-WO-PCT samples were thawed at room temperature, pooled per cow on an equal-weight (of FM) basis, freeze-dried for approximately 96 h in a Zirbus sublimator 3-4-5/20 (Zirbus Technology Benelux B.
• CTX type I collagen
• IDS Plc. Tyne & Wear, UK
• sample Size was based on a two-sided test with a confidence level of 95% and a power of 0.80 to detect a statistically significant difference in ATTD of P in the phytase supplemented treatments compared to CON.
• the expected effect size was based on published data concerning the variance in P digestibility in lactating dairy cows (Valk et al., 2002; Wu et al., 2003; Kincaid et al., 2005; Knowlton et al. 2007).
• the chemical composition of the total rations was calculated based on the chemical composition and intakes of both forages and concentrates.
• the fecal excretion of DM was calculated for each cow from the daily TiO2 administration (g/animal) divided by the TiO2 concentration (g/kg DM) in feces. For this, a fecal recovery of TiO2 of 100% (Glindemann et al., 2009) was assumed.
• the fecal excretion of CP, starch, NDF, P, Ca and PP was calculated as DM fecal excretion multiplied by the concentration of the respective chemical component in the feces.
• the PP level of the total rations was also slightly lower than formulated (-0.2 g/kg DM) but again was similar among treatments (0.6 to 0.7 g/kg DM).
• Phytase activity in the CON concentrate was low (431 FTU/kg, DM basis; Table 2). Because no phytase was added to the CON concentrate, this activity was considered to have been from the intrinsic phytase of the feed ingredients in the concentrate. After accounting for this native phytase activity in the concentrates, the exogenous phytase activities in the total rations were calculated as 1,813 and 6,403 FTU/kg DM in PhyG2,000 and PhyG5,000, respectively (Table 3).
• Example 3 Nutrient intake, excretion, and ATTD The effect of treatment on nutrient intake, ATTD and fecal excretion during the last 5 days of the Exp. is presented in Table 5. There was no effect of treatment on total DMI during this period. Because of this, and the similar chemical composition of the total rations, there was also no effect of treatment on the intake of CP, starch, NDF, total P or Ca.
• A,B Means within a row bearing different superscript upper case letters are different at a statistical tendency level of P ⁇ 0.1.
• the ATTD CP was higher for cows fed PhyG2,000 and PhyG5,000 than CON (+2.7 and +3.8% points, respectively; P ⁇ 0.05) whilst ATTD of P was higher in cows fed PhyG5,000 compared to CON (by 7.8% points; P ⁇ 0.05) and ATTD of Ca tended to be higher in cows fed PhyG5,000 compared to CON (by 9.2% points).
• the increase in ATTD of PP between 0 and 5,000 FTU/kg was 4.6% points.
• cows exhibited a similar DMI, and because the chemical composition was similar between diets, the intake of all chemical components among treatments was also similar, except for PP.
• the PP intake was 12% lower with PhyG5,000, compared to the control diet. This appears to have resulted from the combination of a lower PP content of the concentrate in this treatment (2.33 vs.
• Example 4 Materials and Methods Attorney Docket No. NB42148-WO-PCT This Example describes the materials and methods used to produce the results in Examples 5 and 6. The study was carried out at the Educational and Research Centre of Animal Husbandry, Hofgut Neumühle, Germany, in January 2023, as a joined project with University of Applied Science Bingen.
• Treatment diets comprised of a control diet (CON) formulated without supplemental phytase and two experimental diets based on the CON diet but supplemented with PhyG at a targeted dose level of 2,000 or 5,000 phytase units (FTU) per kilogram of total ration on a dry matter (DM) basis.
• CON control diet
• FTU phytase units
• Treatment diets were provided to cows as a Total Mixed Ration (TMR) containing a forage component comprised of a mixture of grass silage, corn silage and pulp silage and a concentrate component formulated to contain phytate-rich ingredients with low ruminal degradability such as formaldehyde-treated rapeseed meal and hydrothermal pressure-treated sunflower seed meal.
• TMR Total Mixed Ration
• Analyzed chemical composition 1 of the forages I tem, g/kg dry matter Grass Corn Pulp (unless otherwise stated) silage silage silage Dry matter g/kg 248 251 259 Ash 89 38 73 Crude protein 147 68 96 Crude fat 43 32 6 Neutral detergent fibre 586 437 524 Starch 9 271 237 Sugar n.d. n.d. 17 Calcium 4.3 2.5 10.7 Phosphorus (P) 3.5 2.3 0.93 Attorney Docket No. NB42148-WO-PCT Phytate-P 0 0 0 n.d. not determined. The concentrates were manufactured by ABZ Diervoeding (Leusden, the Netherlands) in mash form.
• NB42148-WO-PCT 1 CON control; PhyG2,000, containing PhyG phytase at 2,000 FTU/kg total ration on DM basis; PhyG5,000, containing PhyG phytase at 5,000 FTU/kg total ration on DM basis.
• TMR On a dry matter basis, TMR contains 36.0% of concentrate, 25.3% of corn silage, 19.9% of grass silage, 18.8% of pulp silage.
• the external marker titanium dioxide (TiO 2 ) was added on top of to the concentrates, at a level of 0.7 % in concentrate (as is), 0.2 % in total TMR.
• phytase was added to the concentrate based on the respective dose level. Diets were provided to cows ad libitum, via sensor-controlled feeding troughs (Roughage-Intake Control, Insentec B.V., Marknesse, Netherlands) which measured the individual feed intake per cow per day.
• the TMR was supplied once daily, at 04:45, with a residual of approximately 5%. Feed refusals were removed and weighed daily.
• the TMRs were formulated within the recommendations of the German Society of Nutrition and Physiology (GfE, 2001), except for P which was intentionally below the recommended P requirement (91.5%) in order to facilitate determination of whether the exogenous phytase would improve P digestibility.
• the chemical composition, feed values and phytase activity of the TMRs are shown in Table 8. Table 8. Chemical composition, feed values and phytase activity of the TMRs.
• mice were randomly assigned to treatments.
• the experiment comprised a 14-day pre-period for data collection to facilitate allocation of cows to blocks based on parity, dry matter intake (DMI) and milk production. During this period, milk yield and feed intake data were collected daily and milk composition and analysis were recorded once per week. This was followed by a 19-day experimental period comprising a 14-d diet adaptation phase and 5 days of feces collection.
• DMI dry matter intake
• Cows were milked twice daily starting at 05:00 and 15:30 in a rapid exit milking parlour equipped on one side with a herringbone milking parlor for 8 cows and on the other with a side-by-side milking parlor for 10 cows (GEA Farm Technologies, Bönen, Germany). Milk samples were collected per week from each cow at the Monday afternoon and Tuesday morning milkings. Samples were preserved in bronopol (2-bromo-2-nitropropane-1,3-diol) for later analysis.
• Milk samples were analyzed for fat, protein, urea, lactose and somatic cell count (SCC) by infrared spectrophotometry using a MilkoScan FT6000 (Foss Aalytical A/S, Hiller ⁇ d, Denmark). Two additional milk samples (50 mL) were taken per cow during the last week of the experiment on Monday evening and Tuesday morning, samples pooled (1:1, v:v) and stored at - 20°, until later analysis of total P and calcium content. The samples were analyzed by an accredited laboratory by ⁇ inductively coupled plasma optical emission spectrometry (ICP-OES) according to method VDLUFA III, 10.8.1.2, 2012 (DIN EN ISO 11885:1009-09).
• ICP-OES inductively coupled plasma optical emission spectrometry
• TMR Total daily amounts of TMR offered and refused were recorded on an individual cow basis.
• the daily feed intake was calculated as the difference between the offered and refused amounts.
• Representative samples (500g, fresh matter basis) of each forage (grass, corn, pulp silage) and of the TMRs were collected on days 3, 10, 13 and 14 of the experimental period.
• the forage samples were taken from the fresh cut-surface of the feed silos, pooled and mixed. Samples were vacuum sealed and frozen at -20°C for later analysis.
• Samples of the concentrates Attorney Docket No. NB42148-WO-PCT 500 g were separately taken from the feed silos on days 3 and 13 of the experimental period and every second day of the fecal sampling period. These were vacuum sealed and stored at - 20°C.
• Fecal grab samples ( ⁇ 300–500 g of fecal matter per sample) were collected from all animals twice daily during the last five days of the experiment. Fecal sampling commenced at 09:00 and 13:00 on d 1, at 08:00 and 17:00 on day 2, at 10:00 and 17:00 on day 3, at 06:00 and 20:00 on day 4 and 06:00 and 13:00 on day 5.
• NB42148-WO-PCT Blood Blood samples were taken from each cow from the coccygeal vein on day 2 of the fecal collection period at approximately 10:00. Samples were allowed to clot for 45 min at room temperature ( ⁇ 19°C) and then centrifuged at 1,900 x g for 20 min at 4°C to extract serum. Serum samples were stored at -20°C until analysis for total P. Body weight: Individual body weight (BW) was recorded twice daily directly after each milking. The BW was recorded via automatic weighing scale (GEA Farm Technologies, GmbH, Boenen, Germany). Sample Size, calculations and statistical analysis: The sample size calculation with the software G*Power (Version 3.1.9.7), based on the power-analysis as described by Cohen (1988).
• the Power analysis based on mean values of P digestibility and standard deviation with a confidence level of 95 % and with a power of 0.7 to detect a statistically significant difference between treatments.
• the total sample size for 3 treatments is 46, meaning that replications should include 15 cows per treatment. In case a cow needed to be excluded from the study, e.g. due to illness, it was decided to utilize a replication of 16 cows per treatment group. All data were averaged per cow and trial phase for the statistical analysis.
• the chemical composition of the total rations was calculated based on the chemical composition and intakes of both forages and concentrates.
• the intakes per day of nutrients and chemical components were calculated based on the individual dry matter intake (DMI) of each cow multiplied by the concentration of the respective nutrient or chemical component in the TMR.
• the yield of FPCM (kg/d) was calculated on a 4% fat and 3.3% protein basis.
• the feed efficiency was calculated as FPCM (kg/d) divided by DMI (kg/d).
• Data on somatic cell count (SCC) were log transformed to obtain a normal distribution before statistical analysis. All statistical analyses were performed using JMP (16.1). Data were analyzed by ANOVA to identify treatment effects. Block was included as a random effect. Data were checked for outliers before analysis.
• Example 5 Feed intake, milk yield and composition, and blood analysis The effect of PhyG supplementation on BW, feed intake, feed efficiency, milk yield, milk composition, SCC and blood analytes, with outliers removed from the dataset, is presented in Table 9. Table 9.
• Example 6 Nutrient intake, excretion, and ATTD The effect of treatment on nutrient intake, ATTD and fecal excretion during the 5-d fecal collection period, with outliers removed from the dataset, is presented in Table 10.
• Table 10 Effect of phytase supplementation on nutrient intake, fecal excretion, and apparent total tract digestibility (ATTD) during the 5-d fecal collection period Dietary treatment 1 I tem CON PhyG2,000 PhyG5,000 SEM* P-value Intake Dry matter, kg/d 24.2 25.2 24.3 0.7 0.569 Ash, kg/d 1.7 1.71 1.65 0.05 0.669 Crude protein, kg/d 4.05 3.98 4.06 0.01 0.874 Neutral detergent fiber, kg/d 10.18 10.78 10.14 0.29 0.242 Phosphorus (P), g/d 72.69 75.56 72.92 2.09 0.569 Phytate-P, g/d 20.91 20.85 20.14 0.58 0.599 Fecal exc
• NB42148-WO-PCT 1 CON control; PhyG2,000, containing PhyG phytase at 2,000 FTU/kg; PhyG5,000, containing PhyG phytase at 5,000 FTU/kg.
• a,b Means bearing different superscript letters within a row are significantly different at P ⁇ 0.10.
• Example 7 Comparative phytase activity analysis during passage of the ruminant digestion system Attorney Docket No. NB42148-WO-PCT This example describes an in-vitro study evaluating the effects on phytate hydrolysis occurring during passage of the ruminant digestion system upon addition of various commercial phytases.
• the model (2-step) included simulation of rumen and abomasum compartments under typical conditions found in-vivo.
• the in-vitro system employed was a modification of the Tilley and Terry method from 1963, which was further optimized specifically for evaluation of phytase performance in the rumen by Brask-Pedersen, et al., (2013).
• Ruminal fluid was collected from 3 rumen-cannulated non-lactating dairy cows (Danish Holstein) maintained on a diet based on hay (grass and barley straw) and concentrate feed (oat, barley, soybean meal, rapeseed cake and beet molasses) comprised in a 68 to 32% DM basis ratio. Cows were fed 14h prior to collection. The ruminal fluid including solids was collected from three cows on the morning of use, mixed in the proportion 1:1:1 (% v/v) and maintained at 38 to 39°C (water bath) during transport.
• Ruminal fluid (pH 6.7) was then filtered through cheese cloth and subsequently mixed with a CO2 degassed Bis-Tris buffer (0.036 M Bis-Tris, 0.12 M NaHCO 3 , 0.008 M NaCl, 0.008M KCl, 0.0006M MgCl 2 , 6H 2 O, and 0.0004 M CaCl 2 , 2H2O) solution in the proportion 1:5. Finally, pH was adjusted to 6.2 (addition of 4 M HCl) at 40°C. This solution will be referred to as the rumen fluid mixture. Ground (Retsch mill, 0.7mm sieve) rapeseed meal (0.60% IP6 P) was used as model substrate/feed.
• a CO2 degassed Bis-Tris buffer 0.036 M Bis-Tris, 0.12 M NaHCO 3 , 0.008 M NaCl, 0.008M KCl, 0.0006M MgCl 2 , 6H 2 O, and 0.0004 M CaCl 2 , 2
• Rapeseed in general has low endogenous phytase activity.
• the phytase activity of the specific rapeseed sample employed in this study was not detectable using a modified ISO 30024 method for analysis of phytase in feed (as described in WO2020106796, Example 5).
• PhyG IFF
• Ronozyme® HiPhos 20000GT Novozymes/DSM
• Natuphos® E 10000G (BASF Nutrition) were evaluated along with a negative control containing rumen fluid mixture, but without exogenous phytase added.
• Phytases included in the study are all sold for commercial use in monogastric species. The phytases were dosed based on their phytase units (FTU) with 2FTU/g of substrate. Activities for standardization were obtained using a modified ISO 30024 method for analysis of phytase in products (as described in WO2020106796, Example 3, incorporated herein by reference).
• IP6 High Performance Ion Exchange Chromatography
• An ideal exogenous phytase should be stable in the rumen and more active in the abomasum, in order to breakdown phytate or phytate- protein complex to make these and other (e.g. minerals) nutrients available for animals.
• PhyG is more active in the abomasum and is able to release more P from phytate in this environment, which will lead to better P absorption.. It can be concluded from these results, that PhyG more efficiently degrades IP6 in the abomasum environment at low pH and under presence of pepsin, resulting in greater total IP6 degradation compared to Ronozyme® HiPhos and Natuphos® E.
• PhyG has potential to release more phosphate (resulting in improved phytate P-digestibility) that can be absorbed and utilized by dairy cows, and thereby supports important physiological processes, due to the more complete removal of IP6 eliminating its potential to act as an anti-nutritional factor.
• ISO, 16472 Animal feeding stuffs – Determination of amylase-treated neutral detergent fibre content (aNDF). iso.org/standard/37898.html Attorney Docket No. NB42148-WO-PCT ISO, 2008.
• ISO 13906 Animal feeding stuffs – Determination of acid detergent fibre (ADF) and acid detergent lignin (ADL) contents. iso.org/standard/43032.html ISO, 2015.
• ISO 11085 Cereals, cereals-based products and animal feeding stuffs – Determination of crude fat content by the Randall extraction method. iso.org/standard/63542.html ISO, 2016.

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