CA2465202C - Phytase-containing animal food and method - Google Patents

Abstract

A method is described for improving the nutritional value of a foodstuff comprising a source of myo-inositol hexakisphosphate by feeding the foodstuff in combination with a phytase expressed in yeast. The method comprises the step of feeding the animal the foodstuff in combination with a phytase expressed in yeast wherein the phytase can be selected from the group consisting of AppA1, AppA2 and a site-directed mutant of AppA. The invention also enables reduction of the feed to weight gain ratio and an increase bone mass and mineral content of an animal. A foodstuff and a feed additive comprising AppA2 or a site-directed mutant of AppA are also described.

Description

PHYTASE-CONTAINING ANIMAL FOOD AND METHOD
FIELD OF THE INVENTION
The present invention is related to a method of improving the nutritional value of a foodstuff and to an improved foodstuff. More particularly, the invention relates to a method of improving the nutritional value of a foodstuff comprising myo-inositol hexakisphosphate by feeding the foodstuff to an animal in combination with a phytase expressed in yeast.
BACKGROUND AND SUMMARY OF THE INVENTION
Phytases are myo-inositol hexakisphosphate phosphohydrolases that catalyze the stepwise removal of inorganic orthophosphate from phytate (myo-inositol hexakisphosphate). Phytate is the major storage form of phosphate in plant feeds, including cereals and legumes. Because monogastric animals such as pigs, poultry, and humans have little phytase in their gastrointestinal tracts nearly all of the ingested phytate phosphate is indigestible. Accordingly, these animals require supplementation of their diets with phytase or inorganic phosphate. In contrast, ruminants have microorganisms in the rumen that produce phytases and these animals do not require phytase supplementation of their diets.
The unutilized phytate phosphate in monogastric animals creates additional problems. The unutilized phytate phosphate is excreted in manure and pollutes the environment. Furthermore, in monogastric animals phytate passes largely intact through the upper gastrointestinal tract where it chelates essential minerals (e.g., calcium and zinc), binds amino acids and proteins, and inhibits enzyme activities. Accordingly, phytase supplementation of the diets of monogastric animals not only decreases requirements for supplementation with inorganic phosphate, but also reduces pollution of the environment caused by phytate, diminishes the antinutritional effects of phytate, and increases the nutritional value of the feed.
There are two types of phytases including a 3-phytase (EC.3.1.3.8) which removes phosphate groups at the 1 and 3 positions of the myo-inositol ring, and a 6-phytase (EC.3.1.3.6) which first frees the phosphate at the 6-position of the ring.
Plants usually contain 6-phytases and a broad range of microorganisms, including bacteria, filamentous fungi, and yeasts, produce 3-phytases. Two phytases, phyA and phyB from Aspergillus niger, have been cloned and sequenced. PhyA has been expressed in Aspergillus niger and the recombinant enzyme is available commercially for use in supplementing animal diets.
Phytase genes have also been isolated from Aspergillus terreus, Myceliophthora thermophila, Aspergillus fumigatus, Emericella nidulans, Talaromyces thermophilus, Escherichia coli (appA), and maize. Additionally, phytase enzymes have been isolated and/or purified from Bacillus sp., Enterobacter sp., Klebsiella terrigena, and Aspergillus ficum.
The high cost of phytase production has restricted the use of phytase in the livestock industry as phytase supplements are generally more expensive than the less environmentally desirable inorganic phosphorous supplements. The cost of phytase can be reduced by enhancing production efficiency and/or producing an enzyme with superior activity.
Yeast expression systems can be used to effectively produce enzymes, in part, because yeast are grown in simple and inexpensive media. Additionally, with a proper signal sequence, the expressed enzyme can be secreted into the culture medium for convenient isolation and purification. Some yeast expression systems are also accepted in the food industry as being safe for the production of food products unlike fungal expression systems which may in some cases be unsafe, for example, for human food manufacturing.
Thus, one aspect of this invention is a method of improving the nutritional value of a foodstuff by supplementing the foodstuff with a yeast-expressed phytase with superior capacity to release phosphate from phytate in foodstuffs. The invention is also directed to a foodstuff with improved nutritional value comprising the yeast-expressed phytase. The phytase can be efficiently and inexpensively produced because the yeast-expressed phytase of the present invention is suitable for commercial use in the feed and food industries with minimal processing.
In one embodiment, a method is provided of improving the nutritional value of a foodstuff consumed by a monogastric animal by increasing the bioavailability of phosphate from phytate wherein the foodstuff comprises myo-inositol hexakisphosphate.
The method comprises the step of feeding to the animal the foodstuff in combination with less than 1200 units of a phytase expressed in yeast per kilogram of the foodstuff, wherein the phytase is Escherichia coli-derived AppA2, and wherein the bioavailability of phosphate from phytate is increased by at least 2-fold compared to the bioavailability of phosphate from phytate obtained by feeding the foodstuff in combination with the same units of a phytase expressed in a non-yeast host cell.
In another embodiment, a method is provided of reducing the feed to weight gain ratio of a monogastric animal by feeding the animal a foodstuff wherein the foodstuff comprises myo-inositol hexakisphosphate. The method comprises the step of feeding to the animal the foodstuff in combination with a phytase expressed in yeast, wherein the phytase is selected from the group consisting of Escherichia co/i-derived AppA2 and a site-directed mutant of Escherichia co/i-derived AppA, and wherein the feed to weight gain ratio of the animal is reduced.
In an alternate embodiment, a method of improving the nutritional value of a foodstuff consumed by a monogastric animal by increasing the bone mass and mineral content of the animal wherein the foodstuff comprises myo-inositol hexakisphosphate. The method comprises the step of feeding to the animal the foodstuff in combination with a phytase expressed in yeast wherein the phytase is selected from the group consisting of Escherichia co/i-derived AppA2 and a site-directed mutant of Escherichia co/i-derived AppA, and wherein the bone mass and mineral content of the animal is increased.
In yet another embodiment, a feed additive composition for addition to an animal feed is provided. The feed additive composition comprises a yeast-expressed phytase and a carrier for the phytase wherein the concentration of the phytase in the feed additive composition is greater than the concentration of the phytase in the final feed mixture.
In still another embodiment, a foodstuff is provided. The foodstuff comprises the above-described feed additive composition wherein the concentration of the phytase in the final feed mixture is less than 1200 units of the phytase per kilogram of the final feed mixture.
In another embodiment, a method is provided of improving the nutritional value of a foodstuff consumed by a monogastric animal wherein the foodstuff comprises myo-inositol hexakisphosphate. The method comprises the steps of spray drying a phytase selected from the group consisting of Escherichia co/i-derived AppA2 and a site-directed mutant of Escherichia co/i-derived AppA, mixing the phytase with a carrier for the phytase and, optionally, other ingredients to produce a feed additive composition for supplementing =

a food stuff with the phytase, mixing the feed additive composition with the foodstuff, and feeding the animal the foodstuff supplemented with the feed additive composition.
In an alternate embodiment, a method is provided of improving the nutritional value of a foodstuff consumed by an avian species by increasing the bioavailability of phosphate from phytate wherein the foodstuff comprises myo-inositol hexakisphosphate. The method comprises the step of feeding to the avian species the foodstuff in combination with less than 1200 units of a phytase expressed in yeast per kilogram of the foodstuff, wherein the bioavailability of phosphate from phytate is increased by at least 1.5-fold compared to the bioavailability of phosphate from phytate obtained by feeding to a non-avian species the foodstuff in combination with the phytase expressed in yeast.
In yet another embodiment, a method is provided of reducing the feed to weight gain ratio of an avian species by feeding the avian species a foodstuff wherein the foodstuff comprises myo-inositol hexakisphosphate. The method comprises the step of feeding to the avian species the foodstuff in combination with a phytase expressed in yeast wherein the feed to weight gain ratio of the animal is reduced.
In still another embodiment, a method is provided of improving the nutritional value of a foodstuff consumed by an avian species by increasing the bone mass and mineral content of the avian species wherein the foodstuff comprises myo-inositol hexakisphosphate. The method comprises the step of feeding to the avian species the foodstuff in combination with a phytase expressed in yeast wherein the bone mass and mineral content of the avian species is increased.
In another embodiment, a method is provided of improving the nutritional value of a foodstuff consumed by an avian species wherein the foodstuff comprises myo-inositol hexakisphosphate. The method comprises the step of feeding to the avian species the foodstuff in combination with a phytase expressed in yeast wherein the number of eggs laid and the weight of the eggs laid by the avian species is increased.

-4a-Specific aspects of the invention include:
- a method of improving the nutritional value of a foodstuff consumed by a monogastric animal wherein the foodstuff increases the bone mass and bone mineral content of the animal by increasing the bioavailability of phosphate from phytate wherein the foodstuff comprises myo-inositol hexakisphosphate, the method comprising the step of feeding to the animal the foodstuff in combination with less than 1200 units of an E. coli 6-phytase expressed in yeast per kilogram of the foodstuff and an encapsulating agent, wherein the bioavailability of phosphate from phytate is increased by at least 2-fold compared to the bioavailability of phosphate from phytate obtained by feeding the foodstuff in combination with the same units of a phytase expressed in a non-yeast host cell;
- a method of improving the nutritional value of a foodstuff consumed by a monogastric animal wherein the foodstuff comprises myo-inositol hexakisphosphate, the method comprising the step of feeding to the animal the foodstuff in combination with an E. coli 6-phytase expressed in yeast and an encapsulating agent, wherein the bone mass and mineral content of the animal is increased;
- a method of improving the nutritional value of a foodstuff consumed by a monogastric animal wherein the foodstuff increases the bone mass and bone mineral content of the animal, and wherein the foodstuff comprises myo-inositol hexakisphosphate, the method comprising the steps of: spray drying an E. coli 6-phytase; mixing the encapsulated phytase with a carrier for the phytase and, optionally, other ingredients to produce a feed additive composition for supplementing the foodstuff with the phytase; mixing the feed additive composition with the foodstuff; and feeding the animal the foodstuff supplemented with the feed additive composition;
- a method of improving the nutritional value of a foodstuff consumed by an avian species wherein the foodstuff increases the bone mass and bone mineral content of the avian species by increasing the bioavailability of phosphate from phytate wherein the foodstuff comprises myo-inositol hexakisphosphate, the method comprising the step of feeding to the avian species the foodstuff in combination with less than 1200 units of an a -4b-E. coil 6-phytase expressed in yeast per kilogram of the foodstuff and an encapsulating agent, wherein the bioavailability of phosphate from phytate is increased by at least 1.5-fold compared to the bioavailability of phosphate from phytate obtained by feeding to a non-avian species the foodstuff in combination with the E. coil 6-phytase expressed in yeast;
- a method of improving the nutritional value of a foodstuff consumed by an avian species wherein the foodstuff comprises myo-inositol hexakisphosphate, the method comprising the step of feeding to the avian species the foodstuff in combination with an E. coli 6-phytase expressed in yeast and an encapsulating agent wherein the bone mass and mineral content of the avian species is increased;
- a method of improving the nutritional value of a foodstuff consumed by an avian species wherein the foodstuff increases the bone mass and bone mineral content of the avian species, and wherein the foodstuff comprises myo-inositol hexakisphosphate, the method comprising the step of feeding to the avian species the foodstuff in combination with an E. coil 6-phytase expressed in yeast and an encapsulating agent wherein the number of eggs laid and the weight of the eggs laid by the avian species is increased;
- use of a foodstuff comprising myo-inositol hexakisphosphate in combination with an E. coil 6-phytase expressed in yeast and an encapsulating agent, for the reduction of the feed to weight gain ratio of a mono gastric animal wherein the foodstuff increases the bone mass and bone mineral content of the animal;
- use of a foodstuff comprising myo-inositol hexakisphosphate in combination with an E. coil 6-phytase expressed in yeast and an encapsulating agent, for the reduction of the feed to weight gain ratio of an avian species wherein the foodstuff increases the bone mass and bone mineral content of the avian species;
- a feed additive composition for addition to an animal feed comprising a yeast-expressed E. coil 6-phytase, an encapsulating agent, and a carrier for the phytase, for use to increase the bone mass and bone mineral content of the animal, wherein the --4c-concentration of the phytase in the feed additive composition is greater than the concentration of the phytase in the final feed mixture; and - a foodstuff comprising the feed additive composition as described herein wherein the concentration of the phytase in the final feed mixture is less than 1200 units of the phytase per kilogram of the final feed mixture.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows the amino acid and nucleotide sequences of AppA2.
Fig. 2 shows the amino acid and nucleotide sequences of Mutant U.

Fig. 3 shows the percent increase in bioavailable phosphate in vivo in chickens fed an animal feed supplemented with Natuphos , Mutant U, AppA or AppA2.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method of improving the nutritional value of a foodstuff consumed by an animal wherein the foodstuff comprises myo-inositol hexakisphosphate, the substrate for the phytase enzymes of the invention. The method comprises the step of feeding to an animal the foodstuff in combination with a bacterial phytase expressed in yeast wherein the bioavailability of phosphate from phytate is increased, the feed to weight gain ratio is reduaed, the bone mass and mineral content of the animal is increased or, for avian species, additionally the egg weight or number of eggs laid is increased. The phytase can be selected from the group consisting of Escherichia coli-derived AppA2 and a site-directed mutant of Escherichia coli derived-AppA. In an alternative embodiment, for avian species, the phytase can be any phytase, including phytases selected from the group consisting of Escherichia co/i-derived AppA, Escherichia co/i-derived AppA2, and a site-directed mutant of Escherichia coil-derived AppA. In some embodiments, the bioavailability of phosphate from phytate, the feed to weight gain ratio, and bone mass and mineral content are improved by at least 2-fold, for example, in an avian species, such as poultry, compared to the improvement in nutritional value obtained by feeding the foodstuff in combination with the same weight percent of a bacterial phytase expressed in a non-yeast host cell. The bioavailability of phosphate from phytate is also increased by at least 1.5-fold in porcine species compared to the improvement in nutritional value obtained by feeding the foodstuff in combination with the same weight percent of a bacterial phytase expressed in a non-yeast host cell. Additionally, the bioavailability of phosphate from phytate and the bone mass and mineral content obtained by feeding an avian species the foodstuff in combination with the bacterial phytase expressed in yeast is increased by at least 1.5-fold compared to the bioavailability of phosphate from phytate and the bone mass and mineral content obtained by feeding a non-avian species the foodstuff in combination with the yeast-expressed bacterial phytase.
As used herein "improving nutritional value" or "increased nutritional value" means an improvement in the nutritional value of a foodstuff as reflected by an increase in the bioavailability of phosphate from phytate, a reduction in the feed to weight gain ratio, an increase in bone mass and mineral content, an increase in the bioavailability of inositol from phytate, an increase in the bioavailability from phytate of minerals such as magnesium, manganese, calcium, iron and zinc in an animal fed the foodstuff, or an increase in egg weight or number of eggs laid for an avian species fed the foodstuff (e.g., for laying hens in the first or subsequent round of laying eggs).
As used herein an increase in the "bioavailability of phosphate from phytate" means an increase in availability of phosphate from phytate as reflected by an increase in weight gain or bone ash weight.
As used herein the term "non-yeast host cell" includes a fungal cell.
As used herein, the term "phytase" means an enzyme capable of catalyzing the removal of inorganic phosphate from myo-inositol hexakisphosphate.
As used herein, the term "phytate" means a composition comprising myo-inositol hexakisphosphate.
In accordance with the invention, the feed to weight gain ratio is calculated by dividing weight gain by feed intake. An increase in bone mass or mineral content is reflected by an increase in the dry weight of tibia or fibula bones or by an increase in ash weight.
A variety of phytase genes may be expressed to produce phytase for use in accordance with the invention. Exemplary of genes that can be used in accordance with the invention are phytase genes derived from bacteria, filamentous fungi, plants, and yeast, such as the appA (Gene Bank accession number M58708) and appA2 (Gene Bank accession number 250016) genes derived from Escherichia coli (E. coli) and the phyA and phyB genes derived from the fungus Aspergillus niger, or any site-directed mutant of these genes that retains or has improved myo-inositol hexakisphosphate phosphohydrolase activity.
Phytase genes can be obtained from isolated microorganisms, such as bacteria, fungus, or yeast, that exhibit particularly high phytase activity.
As described below, the appA2 gene was cloned from such an E. coli isolate, and it is exemplary of such a phytase gene.
The expressed phytase gene can be a heterologous gene, or can be a homologous gene. A heterologous gene is defined herein as a gene originating from a different species than the species used for expression of the gene. For example, in the case of expression of a heterologous phytase gene, a phytase gene derived from E.
colt or another species of bacteria can be expressed in a yeast species such as Saccharomyces cerevisiae or Pichia pastoris. A homologous gene is described herein as a gene originating from the same species used for expression of the gene. In the case of expression of a homologous phytase gene, a phytase gene derived from Saccharomyces cerevisiae can be expressed, for example, in the same yeast species.
Exemplary genes for use in producing phytase for use in accordance with the invention are appA, appA2, and site-directed mutants of appA or appA2.
Substituted, deleted, and truncated phytase genes, wherein the resulting expressed phytase, or a fragment thereof, retains substantially the same phytase activity as the phytases specifically exemplified herein, are considered equivalents of the exemplified phytase genes and are within the scope of the present invention.
The appA gene was isolated from E. colt (see U.S. Patent No. 6,451,572.
The appA2 gene was isolated from a bacterial colony that exhibited particularly high phytase activity obtained from the colon contents of crossbred Hampshire-Yorkshire-Duroc pigs (see U.S. Patent Application No.
09/540,149, now U.S. Patent No. 6,511,699). The AppA2 protein product exhibits a pH
optimum between about 2.5 and about 3.5. The amino acid sequence of AppA2 is as shown in SEQ
ID Nos.: 2, 3, and 10. Fig. I shows the amino acid and nucleotide sequences of AppA2.
The untranslated region is indicated by lowercase letters. The underlined sequences are the primers used to amplify appA2 (Pfl: 1-22, and K2: 1468-1490), appA2 (E2: 243-252, and K2: 1468-1490). Potential N-glycosylation sites are boxed. The sequence of appA2 has been transmitted to Genebank data library with accession number 250016. The nucleotide sequence of AppA2 is as shown in SEQ ID No.: 1.
Several site-directed mutants of appA have been isolated (see PCT
Publication No. WO 01/36607 Al (U.S. Patent Application No. 60/166,179.
These mutants were designed to enhance glycosylation of the AppA
enzyme. The mutants include A131NN134N/D207N/S211N, C200N/D207N/S211N
(Mutant U), and A131NN134N/C200N/D207N/S211N (see Rodriguez et al., Arch. of Biochem. and Biophys. 382: 105-112 (2000). Mutant U
has a higher specific activity than AppA, and, like AppA2, has a pH optimum of between about 2.5 and about 3.5. The C200N mutation in Mutant U is in a gapped region and C200 is involved with C210 in forming a unique disulfide bond in AppA. Fig. 2 shows the amino acid and nucleotide sequences of Mutant U. The amino acid sequence of Mutant U
is shown in SEQ ID No.: 5, and the nucleotide sequence of Mutant U is shown in SEQ ID
No.: 4.
Any yeast expression system or other eukaryotic expression system known to those skilled in the art can be used in accordance with the present invention. For example, various yeast expression systems are described in U.S. Patent Application No.
09/104,769 (now U.S. Patent No. 6,451,572), U.S. Patent Application No.
09/540,149 (now U.S. Patent No. 6,511,699), and in U.S. Patent Application No. 60/166,179 (PCT
Publication No. WO 01/36607 Al). Any of these yeast expression systems can be used.
Alternatively, other eukaryotic expression systems can be used such as an insect cell expression system (e.g., SO cells), a fungal cell expression system (e.g., Trichoderma), or a mammalian cell expression system.
A yeast expression system can be used to produce a sufficient amount of the phytase being secreted from the yeast cells so that the phytase can be conveniently isolated and purified from the culture medium. Secretion into=the culture medium is controlled by a signal peptide (e.g., the phyA signal peptide or yeast a-factor signal peptide) capable of directing the expressed phytase out of the yeast cell. Other signal peptides suitable for facilitating secretion of the phytase from yeast cells are known to those skilled in the art.
The signal peptide is typically cleaved from the phytase after secretion.
If a yeast expression system is used, any yeast species suitable for expression of a phytase gene can be used including such yeast species as Saccharomyces species (e.g., Saccharomyces cerevisiae), Kluyveromyces species, Torulaspora species, Schizosaccharomyces species, and methylotrophic yeast species such as Pichia species (e.g., Pichia pastoris), Hansenula species, Torulopsis species, Candida species, and =
Karwinskia species. In one embodiment the phytase gene is expressed in the methylotrophic yeast Pichia pastoris. Methylotrophic yeast are capable of utilizing methanol as a sole carbon source for the production of the energy resources necessary to maintain cellular function, and contain a gene encoding alcohol oxidase for methanol utilization.
Any host-vector system known to the skilled artisan (e.g., a system wherein the vector replicates autonomously or integrates into the host genome) and compatible with yeast or another eukaryotic cell expression system can be used. In one embodiment, the vector has restriction endonuclease cleavage sites for the insertion of DNA
fragments, and genetic markers for selection of transformants. The phytase gene can be functionally linked to a promoter capable of directing the expression of the phytase, for example, in yeast, and, in one embodiment, the phytase gene is spliced in frame with a transcriptional enhancer element and has a terminator sequence for transcription termination (e.g.., HSP150 terminator). The promoter can be a constitutive (e.g., the 3-phospho-glycerate kinase promoter or the a-factor promoter) or an inducible promoter (e.g., the ADH2, GAL-1-10, GAL 7, PH05, T7, or metallothionine promoter). Various host-vector systems are described in U.S. Patent Application No. 09/104,769 (now U.S. patent No.
6,451,572), U.S. Patent Application No. 09/540,149 (now U.S. Patent No. 6,511,699), and in U.S.
Patent Application No.60/166,179 (PCT Publication NO. WO 01/3607 Al).
Yeast cells are transformed with a gene-vector construct comprising a phytase gene operatively coupled to a yeast expression system using procedures known to those skilled in the art. Such transformation protocols include electroporation and protoplast transformation.
The transformed yeast cells may be grown by a variety of techniques including batch and continuous fermentation in a liquid medium or on a semi-solid medium. Culture media for yeast cells are known in the art and are typically supplemented with a carbon source (e.g., glucose). The transformed yeast cells can be grown aerobically at 30 C in a controlled pH environment (a pH of about 6) and with the carbon source (e.g., glucose) maintained continuously at a predetermined level known to support growth of the yeast cells to a desired density within a specific period of time.
The yeast-expressed phytase for use in accordance with the method of the present invention can be produced in purified form by conventional techniques (for example, at least about 60% pure, or at least about 70-80% pure). Typically, the phytase is secreted into the yeast culture medium and is collected from the culture medium. For purification from the culture medium the phytase can, for example, be subjected to ammonium sulfate precipitation followed by DEAE-Sepharose column chromatography.
Other conventional techniques known to those skilled in the art can be used such as gel filtration, ion exchange chromatography, DEAE-Sepharose column chromatography, affinity chromatography, solvent-solvent extraction, ultrafiltration, and HPLC.

Alternatively, purification steps may not be required because the phytase may be present in such high concentrations in the culture medium that the phytase is essentially pure in the culture medium (e.g., 70-80% pure).
In cases where the phytase is not secreted into the culture medium, the yeast cells can be lysed, for example, by sonication, heat, or chemical treatment, and the homogenate centrifuged to remove cell debris. The supernatant can then be subjected to ammonium sulfate precipitation, and additional fractionation techniques as required, such as gel filtration, ion exchange chromatography, DEAE-Sepharose column chromatography, affinity chromatography, solvent-solvent extraction, ultrafiltration, and HPLC
to purify the phytase. It should be understood that the purification methods described above for purification of phytases from the culture medium or from yeast cells are nonlimiting and any purification techniques known to those skilled in the art can be used to purify the yeast-expressed phytase if such techniques are required to obtain a substantially pure phytase.
In one embodiment, the phytase is collected from the culture medium without further purification steps by chilling the yeast culture (e.g., to about 8 C) and removing the yeast cells using such techniques as centrifugation, microfiltration, and rotary vacuum filtration. The phytase in the cell-free medium can be concentrated by such techniques as, for example, ultrafiltration and tangential flow filtration.
Various formulations of the purified phytase preparation may be prepared.
The phytase enzymes can be stabilized through the addition of other proteins (e.g., gelatin and skim milk powder), chemical agents (e.g., glycerol, polyethylene glycol, EDTA, potassium sorbate, sodium benzoate, and reducing agents and aldehydes), polysaccharides, monosaccharides, lipids (hydrogenated vegetable oils), sodium phytate, and other phytate-containing compounds, and the like. Phytase enzyme suspensions can also be dried (e.g., spray drying, drum drying, and lyophilization) and formulated as powders, granules, pills, mineral blocks, liquids, and gels through known processes. Gelling agents such as gelatin, alginate, collagen, agar, pectin and carrageenan can be used. The invention also extends to a feed innoculant preparation comprising lyophilized nonpathogenic yeast which can express the phytases of the present invention in the gastrointestinal tract of the animal when the animal is fed the preparation.
In one embodiment, the phytase in the cell-free culture medium is concentrated such as by ultrafiltration and spray drying of the ultrafiltration retentate. The spray dried powder can be blended directly with a foodstuff, or the spray dried powder can be blended with a carrier for use as a feed additive composition for supplementation of a foodstuff with phytase. In one embodiment, the phytase in the retentate is co-dried with a carrier and/or stabilizer. In another embodiment, the phytase is spray dried with an ingredient that helps the spray dried phytase to adhere to a carrier, or, alternatively, the phytase can loosely associate with the carrier. The feed additive composition (i.e., the phytase/carrier composition and, optionally, other ingredients) can be used for blending with the foodstuff to achieve more even distribution of the phytase in the foodstuff.
Exemplary feed additive compositions (i.e., phytase/carrier compositions and, optionally, other ingredients) can contain 600 units of phytase/gram of the carrier to 5000 units of phytase/gram of the carrier. These phytase/carrier compositions can contain additional ingredients. For example, the compositions can be formulated to contain rice hulls or wheat middlings as a carrier (25-80 weight percent), the phytase (0.5 to 20 weight percent), calcium carbonate (10 to 50 weight percent), and oils (1 to 3 weight percent).
Alternatively, the feed additive composition can include the phytase and the carrier and no additional ingredients. The feed additive composition may be mixed with the feed to obtain a final feed mixture with from about 50 to about 2000 units of phytase/kilogram of the feed.
Thus, a foodstuff comprising a source of myo-inositol hexakisphosphate, a yeast-expressed phytase, and a carrier is also provided in accordance with the invention.
Additionally, a method of improving the nutritional value of a foodstuff consumed by a monogastric animal wherein the foodstuff comprises myo-inositol hexakisphosphate is provided wherein the method comprises the steps of spray drying a phytase, including a phytase selected from the group consisting of Escherichia co/i-derived AppA, Escherichia co/i-derived AppA2, and a site-directed mutant of Escherichia co/i-derived AppA, mixing the phytase with a carrier, and, optionally, other ingredients, to produce a feed additive composition for supplementing a foodstuff with the phytase, mixing the feed additive composition with the foodstuff, and feeding the animal the foodstuff supplemented with the feed additive composition.
In these embodiments, the carrier can be any suitable carrier for making a feed additive composition known in the art including, but not limited to, rice hulls, wheat middlings, a polysaccharide (e.g., specific starches), a monosaccharide, mineral oil, vegetable fat, hydrogenated lipids, calcium carbonate, gelatin, skim milk powder, phytate and other phytate-containing compounds, a base mix, and the like. A base mix typically comprises most of the ingredients, including vitamins and minerals, of a final feed mixture except for the feed blend (e.g., cornmeal and soybean meal). The phytase for use in the feed additive composition is preferably E. co/i-derived AppA, E. co/i-derived AppA2, or a site-directed mutant of E. co/i-derived AppA.
The feed additive composition containing the spray dried phytase and a carrier and, optionally, other ingredients, is mixed with the final feed mixture to obtain a feed with a predetermined number of phytase units/kilogram of the feed (e.g., about 50 to about 2000 units phytase/kilogram of the feed). Before blending with the carrier, the spray dried phytase is assayed for phytase activity to determine the amount of dried powder to be blended with the carrier to obtain a feed additive composition with a predetermined number of phytase units/gram of the carrier. The phytase-containing carrier is then blended with the final feed mixture to obtain a final feed mixture with a predetermined number of phytase units/kilogram of the feed.
Accordingly, the phytase concentration in the feed additive composition is greater than the phytase concentration in the final feed mixture.
In accordance with one embodiment of the invention the foodstuff is fed in combination with the bacterial yeast-expressed phytase to any monogastric animal (i.e., an animal having a stomach with a single compartment). Monogastric animals that can be fed the foodstuff in combination with a yeast-expressed bacterial phytase include agricultural animals, such as porcine species (e.g., barrows (i.e., castrated male gilts (i.e., female pigs prior to first mating) and any other type of swine), chickens, turkeys (poults (i.e., first several weeks post-hatching) and older animals), ducks, and pheasants, any other avian species, marine or fresh water aquatic species, animals held in captivity (e.g., zoo animals), or domestic animals (e.g., canine and feline).
Agricultural monogastric animals are typically fed animal feed compositions comprising plant products which contain phytate (e.g., cornmeal and soybean meal contain phytate (myo-inositol hexakisphosphate)) as the major storage form of phosphate, and, thus, it is advantageous to supplement the feed with phytase. Accordingly, the foodstuffs that can be supplemented with phytase in accordance with the invention include feed for agricultural animals such pig feed and poultry feed, and any foodstuff for avian species or marine or fresh water aquatic species (e.g., fish food). In addition, humans can be fed any foodstuff, such as a cereal product, containing phytate in combination with the yeast-expressed phytase of the present invention.
In the case of an animal feed fed to monogastric animals, any animal feed blend known in the art can be used in accordance with the present invention such as rapeseed meal, cottonseed meal, soybean meal, and cornmeal, but soybean meal and cornmeal are particularly preferred. The animal feed blend is supplemented with the yeast-expressed phytase, but other ingredients can optionally be added to the animal feed blend.
Optional ingredients of the animal feed blend include sugars and complex carbohydrates such as both water-soluble and water-insoluble monosaccharides, disaccharides and polysaccharides. Optional amino acid ingredients that can be added to the feed blend are arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, tyrosine ethyl HC1, alanine, aspartic acid, sodium glutamate, glycine, proline, serine, cysteine ethyl HC1, and analogs, and salts thereof. Vitamins that can be optionally added are thiamine HC1, riboflavin, pyridoxine HC1, niacin, niacinamide, inositol, choline chloride, calcium pantothenate, biotin, folic acid, ascorbic acid, and vitamins A, B, K, D, E, and the like. Minerals, protein ingredients, including protein obtained from meat meal or fish meal, liquid or powdered egg, fish solubles, whey protein concentrate, oils (e.g., soybean oil), cornstarch, calcium, inorganic phosphate, copper sulfate, salt, and limestone can also be added. Any medicament ingredients known in the art can be added to the animal feed blend such as antibiotics.
The feed compositions can also contain enzymes other than the yeast-expressed phytase. Exemplary of such enzymes are proteases, cellulases, xylanases, and acid phosphatases. For example, complete dephosphorylation of phytate may not be achieved by the phytase alone and addition of an acid phosphatase may result in additional phosphate release. A protease (e.g., pepsin) can be added, for example, to cleave the yeast-expressed phytase to enhance the activity of the phytase. Such a protease-treated phytase may exhibit enhanced capacity to increase the bioavailability of phosphate from phytate, to reduce the feed to weight gain ratio, to increase bone mass and mineral content, and to increase the egg weight or number of eggs laid for an avian species compared to intact yeast-expressed phytase. Additionally, combinations of phytases can be used, such as any combinations that may act synergistically to increase the bioavailability of phosphate from phytate, or proteolytic fragments of phytases or combinations of proteolytic fragments can be used. In this regard, the phytase gene expressed in yeast could be used to produce a truncated product directly for use in the method of the present invention.
Antioxidants can also be added to the foodstuff, such as an animal feed composition, to prevent oxidation of the phytase protein used to supplement the foodstuff.
Oxidation can be prevented by the introduction of naturally-occurring antioxidants, such as beta-carotene, vitamin E, vitamin C, and tocopherol or of synthetic antioxidants such as butylated hydroxytoluene, butylated hydroxyanisole, tertiary-butylhydroquinone, propyl gallate or ethoxyquin to the foodstuff. Compounds which act synergistically with antioxidants can also be added such as ascorbic acid, citric acid, and phosphoric acid. The amount of antioxidants incorporated in this manner depends on requirements such as product formulation, shipping conditions, packaging methods, and desired shelf-life.
In accordance with one method of the present invention, the foodstuff, such as an animal feed, is supplemented with amounts of the yeast-expressed phytase sufficient to increase the nutritional value of the foodstuff. For example, in one embodiment, the foodstuff is supplemented with less than 2000 units (U) of the phytase expressed in yeast per kilogram (kg) of the foodstuff. This amount of phytase is equivalent to adding about 34 mg of the phytase to one kg of the foodstuff (about .0034% w/w). In another embodiment, the foodstuff is supplemented with less than 1500 U of the phytase expressed in yeast per kg of the foodstuff. This amount of phytase is equivalent to adding about 26 mg of the phytase to one kg of the foodstuff (about .0026% w/w). In another embodiment, the foodstuff is supplemented with less than 1200 U of the phytase expressed in yeast per kg of the foodstuff. This amount of phytase is equivalent to adding about 17 mg of the phytase to one kg of the foodstuff (about .0017 % w/w). In another embodiment the foodstuff, such as an animal feed composition, is supplemented with about 50 U/kg to about 1000 U/kg of the yeast-expressed phytase (i.e., about 0.7 to about 14.3 mg/kg or about .00007 % to about .0014 % (w/w)). In yet another embodiment the foodstuff is supplemented with about 50 U/kg to about 700 U/kg of the yeast-expressed phytase (i.e., about 0.7 to about 10 mg/kg or about .00007 % to about .001 % (w/w)). In still another embodiment the foodstuff is supplemented with about 50 U/kg to about 500 U/kg of the yeast-expressed phytase (i.e., about 0.7 to about 7 mg/kg or about 0.00007 %
to about .007 % (w/w)). In yet another embodiment, the foodstuff is supplemented with about 50 U/kg to about 200 U/kg of the yeast-expressed phytase (i.e., about 0.7 to about 2.9 mg/kg or about .00007 % to about .0003 % (w/w)). In each of these embodiments it is to be understood that "kg" refers to kilograms of the foodstuff, such as the final feed composition in the case of an animal feed blend (i.e., the feed in the composition as a final mixture). In addition, one unit (U) of phytase activity is defined as the quantity of enzyme required to produce 1 /Imo' of inorganic phosphate per minute from 1.5 mmol/L
of sodium phytate at 37 C and at a pH of 5.5.
The yeast-expressed phytase can be mixed with the foodstuff, such as an animal feed (i.e., the feed composition as a final mixture), prior to feeding the animal the foodstuff or the phytase can be fed to the animal with the foodstuff without prior mixing.
For example, the phytase can be added directly to an untreated, pelletized, or otherwise processed foodstuff, such as an animal feed, or the phytase can be provided separately from the foodstuff in, for example, a mineral block, a pill, a gel formulation, a liquid formulation, or in drinking water. In accordance with the invention, feeding the animal the foodstuff "in combination with" the phytase means feeding the foodstuff mixed with the phytase or feeding the foodstuff and phytase separately without prior mixing.
The yeast expressed-phytase can be in an unencapsulated or an encapsulated form for feeding to the animal or for mixture with an animal feed blend.
Encapsulation protects the phytase from breakdown and/or oxidation prior to ingestion by the animal (i.e., encapsulation increases the stability of the protein) and provides a dry product for easier feeding to the animal or for easier mixing with, for example, an animal feed blend. The yeast-expressed phytase can be protected in this manner, for example, by coating the phytase with another protein or any other substances known in the art to be effective encapsulating agents such as polymers, waxes, fats, and hydrogenated vegetable oils. For example, the phytase can be encapsulated using an art-recognized technique such as a Na2+-alginate encapsulation technique wherein the phytase is coated with Na2+-alginate followed by conversion to Ca2+-alginate in the presence of Ca2+ ions for encapsulation.
Alternatively, the phytase can be encapsulated by an art-recognized technique such as prilling (i.e., atomizing a molten liquid and cooling the droplets to form a bead). For example, the phytase can be prilled in hydrogenated cottonseed flakes or hydrogenated soy bean oil to produce a dry product. The phytase can be used in an entirely unencapsulated form, an entirely encapsulated form, or mixtures of unencapsulated and encapsulated phytase can be added to the foodstuff, such as an animal feed composition, or fed directly to the animal without prior mixing with the foodstuff. Any phytase for use in accordance with the method of the present invention can be similarly treated.
In accordance with the method of the present invention, the phytase-containing foodstuff can be administered to animals orally in a foodstuff, such as an animal feed, or in a mineral block or in drinking water, but any other effective method of administration known to those skilled in the art can be utilized (e.g., a pill form). The foodstuff containing yeast-expressed phytase can be administered to the animals for any time period that is effective to increase the bioavailability of phosphate from phytate, to reduce the feed to weight gain ratio, or to increase the bone mass and mineral content of the animal. For example, in the case of a feed composition fed to a monogastric animal, the feed composition containing yeast-expressed phytase can be fed to the animal daily for the lifetime of the animal. Alternatively, the phytase-containing feed composition can be fed to the animal for a shorter time period. The time periods for feeding the phytase-containing foodstuff to animals are nonlimiting and it should be appreciated that any time period determined to be effective to enhance animal nutrition by administering the phytase-containing foodstuff can be used.

ANIMAL FEED BLEND COMPOSITION
The composition of the animal feed blend for chicks and pigs (i.e., the feed composition without phytase) was as follows:
Table 1. Composition of the animal feed blend used in chick and pig assays.
Ingredient Chick Assays Pig Assay Cornstarch to 100.0 to 100.0 Corn 50.89 61.35 Soybean meal, dehulled 39.69 31.19 Soybean oil 5.00 3.00 Limestone, ground 1.67 1.06 Salt 0.40 Chick vitamin mix 0.20 Pig vitamin mix 0.20 Chick trace mineral mix 0.15 Pig trace vitamin mix 0.35 Choline chloride (60%) 0.20 Pig antibiotic premix (CSP) 0.50 Bacitracin premix 0.05 Copper sulfate 0.08 L-Lysine HC1, feed grade 0.17 DL-Methionine, feed grade 0.20 0.05 PHYTASE PREPARATION
Yeast seed cultures were inoculated in growth medium with Pichia pastoris X33 transformed with either A0X1-appA,pGAP-appA2, or A0X1-Mutant U. The seed cultures were grown at 30 C for about 24 hours until an 0D600 of about 50 was reached.

The seed cultures were then used to inoculate fermentors (batch process) containing sterile FM-22 growth medium containing 5% glucose. The 24-hour seed cultures were diluted about 1:25 to about 1:50 into the FM-22 growth medium.
The yeast cultures were incubated aerobically in the fermentors at 30 C with pH control at 6.0 (using NH2OH) and with continuous glucose feed until the cultures reached an 0D600 of about 400 (about 36 hours).
To collect the phytases from the culture medium, the yeast cultures were rapidly chilled to 8 C. The cells were separated from the culture medium by centrifugation and by microfiltration. The phytases were 70-80% pure in the culture medium and were prepared for blending with a carrier as a feed additive as follows.
The cell-free media containing the secreted phytases were concentrated by ultrafiltration (10,000 MW exclusion limit). The ultraffltration retentates (7-5% solids) were transferred to sterile containers for spray drying. The retentates were spray dried using standard techniques known in the art and the resulting powder was collected (4-6%
moisture).
Microbiological testing of the powder was performed and the powder was assayed for phytase activity. The phytase activity of the powder (units of phytase activity/mg of powder) was used to determine the amount of dried powder to be blended with wheat middlings (i.e., the carrier) to obtain a phytase/carrier mixture with a predetermined number of phytase units/gram of the carrier. The dried phytase powder was mixed with the wheat middlings and packaged in moisture-proof containers. The phytase-containing wheat middlings were mixed with an animal feed blend as needed to obtain a final feed mixture with a predetermined number of phytase units/kg of the feed (about 400 to about 1000 U/kg).

FEED ADDITIVE COMPOSITION
The following compositions are exemplary of feed additive compositions that may be mixed with an animal feed blend, such as the animal feed blend described in Example 1, to obtain a final feed mixture with, for example, about 50 U of phytase/kilogram of the final feed mixture to about 2000 U of phytase/kilogram of the feed.
The feed additive compositions described below are nonlimiting and it should be appreciated that any phytase-containing feed additive composition determined to be effective to enhance the nutritional value of animal feed may be used.
Exemplary feed additive compositions are shown for a feed additive composition containing 600 units of phytase/gram of the feed additive composition or 5000 units of phytase/gram of the feed additive composition.
600 phytase units/gram 5000 phytase units/gram _(weight percent) (weight percent) Rice hulls 82.64 76.35 Calcium carbonate 15.00 15.00 Oil 1.5 1.5 Enzyme 0.86 7.15 600 phytase units/gram 5000 phytase units/gram _fweight percent) (weight percent) Wheat middlings 82.64 76.35 Calcium carbonate 15.00 15.00 Oil 1.5 1.5 Enzyme 0.86 7.15 FEEDING PROTOCOL
Chicks were fed using the protocol described in Biehl, et al. (J. Nutr.
125:2407-2416 (1995)). Briefly, assays were conducted with male and female chicks from the cross of New Hampshire males and Columbian females and were conducted in an environmentally controlled laboratory room with 24 hour fluorescent lighting.
From day 0 to day 7 posthatching, chicks were fed a basal diet of 23% crude protein, methionine-fortified corn-soybean meal as described above in Example 1. On day 8, chicks were weighed, wingbanded and assigned randomly to experimental treatments. Five pens of three or four chicks per pen received each dietary treatment for a 13-day experimental feeding period, and the chicks had an average initial weight of 80 to 100 grams.

Throughout the 13-day feeding period, chicks were confined in thermostatically controlled stainless-steel chick batteries, and stainless-steel feeders and waterers were also used. These steps were taken to avoid mineral contamination from the environment. Diets and distilled deionized water were freely available throughout the feeding period.
Pigs were fasted for 12 hours before the beginning of each assay, were fed the experimental diets for 23 days, and were fasted for 12 hours after each assay was completed. Ten pigs were used per treatment group and the pigs averaged about 8-120 kg at the initiation of the assay. Pigs were housed in individual pens that contained a stainless-steel feeder, a stainless-steel waterer, and galvanized round-bar fencing.
All of the chicks in each treatment group and the five median-weight pigs of each treatment group were euthanized for testing. Body weight gain was measured and tibia (chicks) or fibula (pigs) bones were harvested for bone ash analysis as a reflection of bone mass and mineral content.

MEASUREMENT OF INORGANIC PHOSPHATE AND BIOAVAILABLE
PHOSPHATE
Total phosphate in the feed samples used to generate a standard curve was quantified colorimetrically according to AOAC (1984) as described in Biehl et al.
Monobasic potassium phosphate (ICH2PO4) served as the standard. A standard curve was generated by measuring inorganic phosphate levels in basal feed supplemented with KH2PO4 (X-axis) and determining tibia ash weight (mg) or weight gain (g) (Y-axis) for animals fed basal feed supplemented with various levels of KH2PO4. The bioavailability of phosphate from phytate was then determined for animals fed basal feed supplemented with phytase by comparison of tibia ash weight and weight gain in these animals to the standard curve.

BONE ASH ANALYSIS
At the end of each experiment, chicks or pigs were euthanized, and right tibia or fibula bones were removed quantitatively from chicks or pigs, respectively. The bones were pooled by replicate pen and, after removal of adhering tissue, were dried for 24 hours at 100 C and were weighed. After weighing, the bones were dry ashed for 24 hours at 600 C in a muffle furnace. Ash weight was expressed as a percentage of dry bone weight and also as ash weight per bone.

PHYTASE EXPRESSION IN YEAST
In accordance with the present invention, any phytase gene may be expressed in yeast, and any yeast expression system may be used according to methods (now U.S. Patent No. 6,511,699), and in U.S. Patent Application No. 60/166,179 (PCT
Expression of the appA gene in Saccharotnyces cerevisiae.
The appA gene was expressed in Saccharomyces cerevisiae linked to the signal peptide of the phyA gene (phytase gene from Aspergillus niger). The appA gene was for 25 cycles with 1 minute of denaturation at 95 C, 1 minute of annealing at 58 C, and 1 minute of chain extension at 72 C.
A 1.3 kb fragment was amplified by PCR, and was digested with Kpnl and EcoRI and ligated into pYES2, a vector for expression in Saccharomyces cerevisiae. The pYES2-appA-phyA signal peptide construct was transformed into the yeast (INVScI, Invitrogen, San Diego, CA) by the lithium acetate method.
Selected transformants were inoculated into YEPD medium and expression was induced with galactose after an 0D600 of 2 was reached. The cells were harvested 15-20 hours after induction. The AppA phytase enzyme was isolated from the culture supernatant and was the major protein present eliminating the need for a tedious purification.
Expression of the appA or appA2 gene in Pichia pastoris.
appA. The template for the PCR reaction was as described above. The 5' primer used for the PCR reaction was as follows: 5' GGA ATT CCA GAG TGA GCC
GGA 3' (SEQ ID No.: 8). The 3' primer was as follows: 5' GGG GTA CCT TAC AAA
CTO CAC G 3' (SEQ ID No.: 9). The amplification reaction included 1 cycle at 94 C (3 mm.), 30 cycles at 94 C (0.8 min), 30 cycles at 54 C (1 min.), 30 cycles at 72 C (2 mm.), and 1 cycle at 72 C (10 min). The product was first inserted into the pGEM T-easy vector (Promega), and E. coli strain TOP1OF' was used as the host to amplify the construct. The construct was then inserted into the yeast expression vector pPIcZaA
(Invitrogen) at the EcoRI site, and E. coli strain TOP1OF' was again used as the host to amplify the construct.
The PIcZa vector containing appA was transformed into Pichia pastoris strain X33 by electroporation. The transformed cells were plated into YPD-Zeocin agar medium and positive colonies were incubated in minimal media with glycerol (BMGY) for 24 hours. When an 0D600 of 5 was reached, the cells were centrifuged and were resuspended in 0.5% methanol medium (BMMY) for induction. Methanol (100%) was added every 24 hours to maintain a concentration of 0.5-1%. The cells were harvested at 192 hours after induction and the AppA protein was purified by ammonium sulfate precipitation and DEAE-Sepharose column chromatography.
appA2. The appA2 gene was isolated (see U.S. Patent Application No.
09/540,149, now U.S. Patent No. 6,511,699) from a bacterial colony that exhibited particularly high phytase activity obtained from the colon contents of crossbred Hampshire-Yorkshire-Duroc pigs.
To isolate a bacterial colony exhibiting high phytase activity the colon contents sample was diluted in an anaerobic rumen fluid glucose medium, was shaken vigorously for 3 minutes, and was serially diluted. The diluted samples were cultured at 37 C for 3 days on a modified rumen fluid-glucose-cellobiose-Agar medium containing insoluble calcium phytate. Colonies with a clear zone were assayed for phytase activity using sodium phytate as a substrate. The colony identified as producing the highest phytase activity was identified as an E. coil strain. Accordingly, the appA2 gene was isolated using the primers as described above for appA expression in Pichia pastoris (SEQ. ID Nos. 8 and 9). The appA2 gene was cloned into the PIcZa vector and Pichia pastoris strain X33 was transformed with the PlcZa-appA2 construct as described above for appA
expression in Pichia pastoris. The AppA2 enzyme was expressed as described above for AppA, and the AppA2 protein was collected from the yeast culture supernatant.
AppA Site-Directed Mutants.
Site-directed mutants of appA were prepared as described in U.S. Patent Application No. 06/166,179 (PCT Publication No. WO 01/36607 Al).
Briefly, the E. coil appA mutants were constructed using the megaprimer site-directed mutagenesis method (Seraphin, B. et al., Nucleic Acids Res.

24;3276-77 (1996); Smith, A.M. eta]., Biotechniques 22: 438-39 (1997).
The template for mutagenesis was obtained from ATCC, and the gene (1.3 kb) was transformed into E. coli strain BL21 (No. 87441) using the pappA I
expression vector (Ostanin et al., J. Biol. Chem., 267:22830-36 (1992)). The template was amplified as described above for appA expressed in Pichia pastoris using the primers used above for appA expression in Pichia pastoris (SEQ. ID Nos.: 8 and 9). The amplification reaction included 1 cycle at 94 C (3 min.), 30 cycles at 94 C (0.5 min), 30 cycles at 54 C (1 min.), cycles at 72 C (1.5 min.), and 1 cycle at 72 C (10 min).
The mutagenesis PCR reaction was performed as described above using the primers as follows:

5'CTGGGTATGGTTGGTTATATTACAGTCAGGT3' Al 31N
(SEQ ID No.: 10) V134N
5'CAAACTTGAACCTTAAACGTGAG3' C200N
(SEQ ID No.: 11) 5'CCTGCGTTAAGTTACAGCTTTCATTCTGTTT3' D207N
(SEQ ID No.: 12) S211N
The mutagenic PCR reactions incorporated appropriate primers to make the A131N/V134N/D207N/S211N, C200N/D207N/S211N (Mutant U), and A131N/V134N/C200N/D207N/S211N mutants of appA. The first mutagenic PCR
reaction (100 1) was performed as described above, using 4 id of the intact appA PCR
reaction mixture and the appropriate modified primers listed above. All megaprimer PCR
products were resolved in a 1.5% low melting agarose gel. The expected fragments were excised and eluted with a GENECLEAN II kit. The final mutagenic PCR reaction (1000) was set up as described above, using 4 Al of the appA PCR product and varying concentrations of the purified megaprimer (50 ng to 4 g), depending on its size. Five thermal cycles were set up at 94 C for 1 minute and 70 C for 2 minutes. While at 70 C, 1 ttmol of forward primer and 2 U of AmpliTaq DNA polymerase were added and gently mixed with the reaction mixture, and thermal cycling continued for 25 cycles at 94 C for 1 minute and 70 C for 1.5 minutes.
The genes encoding the site-directed mutants were expressed in Pichia pastoris as described above for the appA2 gene. The protein products were expressed as described above for AppA, and the site-directed mutants were purified from the yeast culture supernatant by ammonium sulfate precipitation and DEAE-Sepharose chromatography.

IN VIVO EFFECTS OF YEAST - EXPRESSED
PHYTASES FED TO CHICKS
To evaluate their potential as animal feed supplements, the yeast-expressed phytases AppA and AppA2, were dried and added to the animal feed blend (23%
feed compositions as described above in Example 4. The treatment groups included various level of KH2PO4 to construct the standard curve, 500 U/kg of Natuphos , a commercially available (Gist-Brocades) phytase expressed in the fungus Aspergillus niger, 500 U/kg of AppA expressed in Pichia pastoris or in E. colt, and various levels of AppA2/p (AppA2 expressed in Pichia pastoris using the constitutive pGAP
promoter for gene expression) as follows:
Treatment Groups:
1. Basal Diet (0.10% P, 0.75% Ca) 2. Same as 1 + 0.05% P from KH2PO4 3. Same as 1 + 0.10% P from KH2PO4 4. Same as 1 + 0.15% P from K.H2PO4 5. Same as 1 + 500 U/kg AppA (yeast) 6. Same as 1 + 500 U/kg AppA (E. colt) 7. Same as 1 + 500 U/kg AppA2/p 8. Same as 1 + 1000 U/kg AppA2/p 9. Same as 1 + 1500 U/kg AppA2/p 10. Same as 1 + 500 U/kg Natuphos For the various treatment groups weight gain, feed intake, the feed to weight gain ratio, dry tibia weight, tibia ash weight, tibia ash weight as a percent of dry tibia weight, and the percentage of bioavailable phosphate based on both tibia ash weight and weight gain were determined. The results are expressed below as a mean for the four chicks for each of the five pens (R1, R2, R3, R4, and R5), and the mean for the five pens was also calculated (labeled "mean" in the tables). The treatment groups are labeled T1-T10 in the tables, and "g/c/d" indicates weight gain or feed intake in grams/chick/day.

Weight gain (We) Ti 12 T3 T4 T5 T6 T7 18 19 110 _Rl 185 282 315 321 314 284 334 . 352 334 R5 223 278 303 332 316 . 268 313 336 361 Mean 219g 283a 314' 327b 317c 299d 321b 335a 344a 276f g/c/d 16.8 21.8 24.2 25.2 24.4 23.0 24.7 25.8 26.5 21.2 Pooled SEM = 6 LSD= 16 13-d Feed intake (g/c) Ti 12 13 T4 T5 T6 17 . T8 19 110 R1 303 . 392 434 434 426 389 450 . 474 465 397 R3 336 391 445 458 446 . 425 428 . 445 464 397 R5 335 388 421 467 425 . 389 453 461 483 420 Mean 331f 410a 436' 4.47abc 4- -.J.zc 411d 439b 459a 467' 399e Weld 25.5 31.5 33.5 34.4 33.2 31.6 33.8 35.3 35.9 30.7 Pooled SEM = 7 LSD = 21 Gain/feed (g/kg) Ti T2 T3 T4 T5 T6 17 T8 T9 110 R3 696 708 736 730 719 736 743 722 736 .

R4 668 700 715 733 . 731 733 738 738 R5 665 717 721 710 742 688 691 730 749 .

Mean 661c 692b 720a 7311 734a 727a 731' 732a 737a 691b Pooled SEM = 10 LSD = 28 Dry tibia weight (mg/c) R1 659 804 . 883 981 892 787 914 1059 1106 R5 714 714 . 866 942 841 809 931 1036 1132 Mean 698g 756f 890d 967c 873d 829e 909d 1028 1096a 747f Pooled SEM = 16 LSD = 45 Tibia Ash (mg/c) Ti T2 T3 14 T5 T6 T7 T8 T9 T10 Mean 237h 299g 413' 490a 428d 381f 447d 559b 616a 290g Pooled SEM = 10 LSD = 28 Supplemental P Intake (g) Ti T2 13 14 R1 0 0.196 0.434 0.651 R2 0 0.231 0.448 0.680 R3 0 0.196 0.445 0.687 R4 0 0.208 0.432 0.636 R5 0 0.194 0.421 0.701 Mean Od 0.205' 0.436h 0.671a Pooled SEM = 0.007 LSD = 0.022 Tibia ash (%) Ti T2 T3 14 T5 16 T7 T8 T9 T10 R1 35.15 38.22 46.03 50.12 48.64 43.78 47.98 55.64 55.79 36.78 R2 32.86 40.92 46.53 51.86 49.06 47.31 48.07 54.69 55.35 40.15 R3 36.30 39.96 46.22 51.61 48.31 46.62 49.85 54.23 56.69 39.14 R4 32.01 40.02 47.47 49.96 47.70 45.96 49.19 54.44 56.23 36.68 R5 33.45 38.82 45.74 49.95 51.40 46.11 50.54 52.86 56.71 41.32 Mean 33.958 39.59f 46.40e 50.70' 49.02d 45.96' 49.13cd 54.37b 56.15' 38.81f Pooled SEM = 0.57 LSD= 1.62 Phosphorus Equivalency Estimates Tibia Ash Weight KH2PO4 Standard Curve: Y = tibia ash (mg) X = supplemental or equivalent P intake (g) Y=232.0 + 389.9X
r2=0.97 For 500 U/kg Phytase activity (example calculations using tibia ash treatment means) % Bioavailable P
AppA (yeast): (428 -232.0)/389.9=0.503 g P from 432 g Fl = 0.116%
AppA (E. coli): (381-232.0)/389.9=0.382 g P from 411 g Fl = 0.093%
AppA2/p: (447-232.0)/389.9=0.551 g P from 439 g Fl = 0.126%
Natuphose: (290-232.0)/389.9=0.149 g P from 399 g Fl = 0.037%
** Results from ANOVA (calculation performed for each pen of four birds;
treatment legend on previous page) Bioavailable P (%) R1 0.122 0.075 0.118 0.194 0.212 0.030 R2 0.127 0.121 0.113 0.179 0.200 0.047 R3 0.117 0.091 0.131 0.168 0.206 0.034 R4 0.095 0.085 0.129 0.198 0.219 0.023 R5 0.121 0.093 0.135 0.176 0.218 0.051 Mean 0.116' 0.093d 0.125c 0.183b 0.211a 0.037e Pooled SEM = 0.005 LSD = 0.016 Contrasts Significance (P-value) AppA (yeast) vs. AppA (E. coli) 0.006 AppA2/p linear 0.001 AppA2/p quadratic 0.039 Weight Gain KH2PO4 Standard Curve: X = weight gain (g) Y = supplemental P intake (g) Y=234.1 + 157.2X
r2=0.84 Results from ANOVA (calculation performed for each pen of four birds;
treatment legend on previous page) Bioavailable P (%) R1 0.119 0.082 0.141 0.158 0.137 0.056 R2 0.123 0.130 0.121 0.130 0.154 0.064 R3 0.124 0.117 0.125 0.124 0.148 0.053 R4 0.121 0.112 0.133 0.148 0.140 0.069 R5 0.123 0.055 0.111 0.141 0.167 0.091 Mean 0.122b 0.099d 0.126b 0.140ab 0.149a 0.067d Pooled SEM = 0.007 LSD = 0.021 Contrasts Significance (P-value) AppA (yeast) vs. AppA (E. coli) 0.038 AppA2/p linear 0.036 AppA2/p quadratic 0.768 Supplementation of the animal feed blend with increasing amounts of KH2PO4 resulted in linear (p<.001) increases in weight gain and tibia ash.
Supplementation of the animal feed blend with Natuphos resulted in linear increases (p<.001) in weight gain, tibia ash, and % bioavailable phosphate. At 500 U/kg the yeast-expressed enzymes (AppA and AppA2/p) were more effective than E. co/i-expressed AppA or Natuphos at improving each of the in vivo responses tested, including the feed to weight gain ratio, tibia weight, and % bioavailable phosphate. In fact, AppA and AppA2/p were 2-6 times more effective at increasing the level of bioavailable phosphate than Natuphos , depending on whether tibia ash weight or weight gain was used to calculate the percent of bioavailable phosphate.

IN VIVO EFFECTS OF YEAST-EXPRESSED
PHYTASES FED TO CHICKS
The procedure was as described in Example 8 except that the chicks had an average initial weight of 91 grams, and the treatment groups were as follows:
Treatment Groups:
1. Basal Diet (0.10% P, 0.75% Ca) 2. Same as 1 + 0.05% P from KH2PO4 3. Same as 1 + 0.10% P from KH2PO4 4. Same as 1 + 300 U/kg Natuphos phytase 5. Same as 1 + 500 U/kg Natuphos phytase 6. Same as 1 + 700 U/kg Natuphos phytase 7. Same as 1 + 900 U/kg Natuphos phytase 8. Same as 1 + 1100 U/kg Natuphos phytase 9. Same as 1 + 1300 U/kg Natuphos phytase 10. Same as 1 + 1500 U/kg Natuphos phytase 11. Same as 1 + 500 U/kg Ronozyme phytase 12. Same as 1 + 300 U/kg Mutant U phytase 13. Same as 1 + 500 U/kg Mutant U phytase 14. Same as 1 + 500 U/kg AppA phytase 15. Same as 1 + 500 U/kg AppA2 phytase The Ronozyme (Roche) phytase is a phytase expressed in fungus. Mutant U is the site-directed mutant of AppA described above. The tables are labeled as described in Example 8. The in vivo effects of phytase supplementation described in Example 8 were measured and the results were as follows:

=
Weight gain (g/c) = Mean 259 290 323 278 289 278 302 295 304 317 266 314 339 327 336 g/c/d 18.5 20.7 23.1 19.9 20.6 19.9 21.6 21.1 21.7 22.6 19.0 22.4 24.2 23.4 24.0 0 Pooled SEM = 6 = LSD = 18 1.) Feed intake (g/c) u.1.) Ti T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 o co Mean 420 443 506 427 433 438 458 459 460 484 424 484 499 487 513 g/c/d 30.0 31.6 36.1 30.5 30.9 31.3 32.7 32.8 32.9 34.6 30.3 34.6 35.6 34.8 36.6 Pooled SEM =10 LSD =27 C--;
CA

Gain/feed (g/kg) Ti T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 Mean 617 654 639 651 666 635 658 643 661 656 627 649 678 672 656 (5) Pooled SEM = 10 LSD = 28 Dry tibia weight (mg/c) 1-d Mean 940 965 1134 964 976 989 1021 1006 1032 1109- 928 1092 1221 1157 1186 Pooled SEM = 22 LSD = 61 c7, Supplemental P intake (g) o --.1 R1 0 0.225 0.489 o R2 0 0.220 0.565.
R3 0 0.223 0.526 R4 0 0.219 0.472 R5 0 0.222 0.478 Mean 0' 0.222b 0.506' Tibia ash (mg/c) 12 13 14 15 n = .

I.) 226 355 481 441 470 a, c7, in 293 410 479 420 455 "

I.) 264 406 500 447 413 I.) .

tal a, I
Mean 264 311 414 289 293 302 327 326 349 373 266 385 467 437 . 448 0 a, Pooled SEM = 10 LSD = 28 co Tibia ash ( /0) R1 29.30 32.94 38.15 29.25 29.48 30.18 32.55 32.54 33.10 32.60 --- 34.90 36.63 39.07 37.06 R2 29.29 30.97 38.03 30.61 30.99 32.06 33.21 32.73 35.51 33.67 27.33 35.66 38.48 39.04 38.89 R3 28.03 31.08 34.70 30.81 29.79 28.71 31.12 33.26 33.45 35.49 28.50 35.63 38.73 35.63 37.48 Iv n R4 26.30 32.33 35.34 29.35 31.17 30.80 31.73 31.55 33.71 33.11 28.74 36.38 39.29 38.00 36.63 Lt.
R5 27.52 33.76 35.98 30.13 28.60 30.81 31.44 31.67 33.09 33.41 .29.77 33.81 38.14 37.18 38.70 cp o Mean 28.09 32.21 36.44 30.03 30.00 30.51 32.01 32.35 33.77 33.65 28.58 35.28 38.25 37.78 37.75 kL.) c.:.) Pooled SEM = 0.49 .6.
o c:
LSD = 1.39 c,.) Phosphorus Equivalency Estimates KH2PO4 Standard Curve:. Y = tibia ash (mg) -:-x = supplemental or equivalent P intake (g) Y = 257.1 + 299.0X
r2 = 0.88 For 500 U/kg Phytase activity (example calculations using tibia ash treatment mean) % Bioavailable P
Natuphosg: (293 - 257.1)/299.0 = 0.120 g P from 433 g FT = 0.030%
Ronozyme0: (266 - 257.1)/299.0 = 0.030 g P from 424 g FT = 0.007%
Mutant U: (467 - 257.1)/299.0 = 0.702 g P from 499 g FT =
0.141% 0 AppA: (437 - 257.1)/299.0 = 0.602 g P from 487 g FT = 0.124%
AppA2: (448 - 257.1)/299.0 = 0.638 g P from 513 g FI = 0.124%

Results from ANOVA (calculation performed for each pen of four birds;
treatment legend on previous page) Bioavailable P (/0) 15 1.) co R1 0.017 0.034 0.037 0.051 0.047 0.062 0.076 --- 0.097 0.117 0.136 0.133 R2 0.031 0.026 0.058 0.057 0.049 0.086 0.072 -0.026 0.071 0.145 0.134 0.137 0.034 0.017 0.005 0.052 0.069 0.061 0.101 0.028 0.098 0.150 0.105 0.124 0.020 0.043 0.030 0.060 0.045 0.053 0.071 0.006 0.104 0.163 0.128 0.106 0.024 0.018 0.040 0.034 0.038 0.071, 0.082 0.017 0.070 0.128 0.117 0.120 Mean 0.025 0.027 0.034 0.051 0.050 0.066 0.080 0.006 0.088 0.140 0.124 0.124 Pooled SEM = 0.006 LSD = 0.018 tµ.) =

Contrasts Significance (P-value) Linear response to Natuphos0 (treatment groups 5 (trt) 4-10) 0.001 Quadratic response to Natuphos 0.208 500 U/kg Natuphos0 (trt 5) vs 500 U/kg yeast-expressed phytases (trt 13-15) 0.001 500 U/kg Natuphose (trt 5) vs 500 U/kg Ronozyme0 (trt 11) 0.031 500 U/kg Ronozymee (trt 11) vs 500 U/kg yeast-expressed phytases (trt 13-15) 0.001 300 U/kg Mutant U (trt 12) vs 500 U/kg Mutant U (trt 13) 0.001 500 U/kg Mutant U (trt 12) vs 500 U/kg AppA (trt 14) 0.074 500 U/kg Mutant U (trt 12) vs 500 U/kg AppA2 (trt 15) 0.074 (5) Multiple Linear Regression: Y =-- tibia ash (mg) X = phytase intake (U) Y = 263.462 + 0.144(Natuphos0) + 0.014(Ronozyrne0) + 0.823(MutantU) +
0.711(AppA) + 0.718(AppA2) R2 = 0.93 Relative Phytase Activity Ratio (IV) Eq. To 500 U/kg Natuphose Ronozyme : (0.014/0.144)*100 = 10 50 Mutant U: (0.823/0.144)*100 = 572 2860 AppA: (0.711/0.144)*100 = 494 2470 AppA2: (0.718/0.144)*100 = 499 2495 1-d =

At 500 U/kg, the yeast-expressed enzymes (Mutant U, AppA and AppA2) were more effective than Natuphos or Ronozyme0 (both enzymes are expressed in fungal expression systems) at improving the in vivo responses tested.
For example, Mutant U, AppA and AppA2 were four times more effective than Natuphose in releasing phosphate (see Fig. 3).

IN VIVO EFFECTS OF YEAST-EXPRESSED
PHYTASES FED TO PIGS
The procedure was as described in Example 8 except that pigs (average initial weight of 10 kg) were fed the phytase-supplemented feed composition.
The treatment groups were as follows:
Treatment Groups:
1) Basal diet (0.75 P; 0.60% Ca) 2) Same as 1 + 0.05% P from KH2PO4 3) Same as 1 + 0.10% P from KH2PO4 4) Same as 1 + 0.15% P from KH2PO4 5) Same as 1 + 400 U/kg phytase from Natuphos0 6) Same as 1 + 400 U/kg phytase from Mutant U phytase 7) Same as 1 + 400 U/kg AppA phytase 8) Same as 1 + 400 U/kg AppA2 phytase For the various treatment groups weight gain, feed to weight gain ratio, fibula ash weight, fibula ash weight as a percentage of dry fibula weight, and the percentage of bioavailable phosphate based on fibula ash weight were determined.
The results were as follows:

Table 3. Pig Assaya Fibula Composition Weight G:F, Ash, Ash, Bioavailable Treatment Groups gain, g/d g/kg mg p, %b Basal Diet 369 533 29.31 666 Same as 1 + 0.05% P from KH2PO4 435 576 32.83 766 Same as 1 + 0.10% P from KH2PO4 446 618 36.62 972 Same as 1 + 0.15% P from KH2PO4 509 660 36.57 1123 Same as 1 + 400 U/kg Natuphos phytase 460 605 34.37 889 0.081 c7, Same as 1 + 400 U/kg Mutant U phytase 458 645 35.45 961 0.116 v:) Same as 1 + 400 U/kg AppA phytase 458 606 35.97 ¨ 1035 0.136 0 Same as 1 + 400 U/kg AppA2 phytase 443 583 34.96 968 0.108 0 Contrast Significance (P-value) Natuphos (treatment group (trt) 5) vs. yeast-expressed NS NS NS
0.05 0.048 phytases (trt 6-8) =
Mutant U (trt 6) AppA vs. (trt 7) and AppA2 (trt 8) 0.10 0.10 NS
0.001 0.239 a Data are means of ten replicates per treatment of individually housed pigs during a period of 23 days; average initial weight was 8.4 0.2 kg. 1-3 Percent bioavailable P calculations are estimates of P equivalency based on KT-12PO4 standard curve (treatments 1-4). Calculations based on KH2PO4 standard curve where Y=fibula ash (mg) and X=supplemental or equivalent P intake (g): Y=664.49 + 15.29X (r2=0.87).

At 400 U/kg, the yeast-expressed enzymes (Mutant U, AppA, and AppA2) were more effective than Natuphos (expressed in fungus) at improving the responses tested.

IN VIVO EFFECTS OF YEAST-EXPRESSED
PHYTASES IN CHICKS
The procedure was as described in Example 8 except that the chicks had an average initial weight of 83 grams, and the treatment groups were as follows:
TREATMENT GROUPS:
1. Basal Diet (0.10% P; 0.75% Ca) 2. Same as 1 + 0.05% P from KH2PO4 3. Same as 1 + 0.10% P from KH2PO4 4. Same as 1 + 0.15% P from KH2PO4 5. Same as 1 + 500 U/kg Natuphos phytase (batch 1) 6. Same as 1 + 500 U/kg Natuphos phytase (batch 2) 7. Same as 1 + 1000 U/kg Natuphos phytase (batch 2) 8. Same as 1 + 500 U/kg Ronozyme phytase (batch 1) 9. Same as 1 + 500 U/kg Ronozyme phytase (batch 2) 10. Same as 1 + 1000 U/kg Ronozyme phytase (batch 2) 11. Same as 1 + 500 U/kg Mutant U phytase 12. Same as 1 + 500 U/kg AppA phytase 13. Same as 1 + 500 U/kg AppA2 phytase 14. Same as 1 + 500 U/kg AppA2 + novel promoter phytase (AppA2/p) The tables are as labeled in Example 8. The in vivo effects of phytase supplementation described in Example 8 were measured and the results were as follows:

Weight gain (g/c) Ti T2 T3 T4 TS T6 T7 T8 T9 T10 T11 T12 T13 T14 Mean 176 253 293 333 218 236 265 224 219 245 319 312 314 318 g/c/d 12.6 18.1 20.9 23.8 15.6 16.9 18.9 16.0 15.6 17.5 22.8 22.3 22.4 22.7 (5) Pooled SEM = 7 LSD = 19 Feed intake (g/c) Ti T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 co Mean 308 372 417 455 351 373 393 349 326 361 429 444 437 449 -g/c/d 22.0 26.6 29.8 32.5 25.1 26.6 ' 28.1 24.9 23.3 25.8 30.6 31.7 31.2 32.1 Pooled SEM = 10 LSD = 28 Gain/feed (g/kg) Ti T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 R5 551 685 690 737 565 622 641 649 624 658 . 721 Mean 569 680 703 731 620 632 675 642 634 682 745 702 718 708 (5) Pooled SEM =13 "
LSD = 37 Dry tibia weight (mg/c) 12 13 14 co 1-d Mean 737 903 1011 1219 823 859 908 832 826 879 1118 1086 1081 1120 Pooled SEM = 27 LSD = 77 Supplemental P intake (g) 1 2 3 4 o R1 0 0.187 0.431 0.636 'a --.1 1--, o R2 0 0.176 0.407 0.661 w R3 0 0.187 0.419 0.708 R4 0 0.193 0.400 0.685 R5 0 0.187 0.427 0.724 Mean Od 0.186` 0.417b 0.683' Tibia ash (mg/c) 11 , 12 13 14 n I.) 395 425 357 353 a, (5) in 410 370 389 454 I.) I.) 416 455 371 396 4.
t...) I.) a, I
Mean 183 272 347 455 224 236 262 227 223 242 404 403 385 409 0 a, I
Pooled SEM = 12 I.) LSD = 32 co Tibia ash (%) R1 25.88 28.82 36.31 36.67 27.60 27.39 26.91 = 29.01 28.42 26.99 37.15 . 37.03 38.24 37.64 R2 24.20 --- 34.21 37.18 26.51 25.89 26.86 27.97 , 26.44 27.16 35.49 36.90 34.33 32.98 R3 25.43 31.61 34.87 37.53 28.06 27.75 29.52 26.65 26.85 28.35 35.54 36.34 34.40 37.22 R4 25.04 31.53 33.85 37.22 27.42 29.15 30.73 26.46 26.03 27.97 35.76 39.09 35.43 36.71 1-d n R5 23.72 28.11 32.27 38.02 26.61 27.17 30.41 26.26 26.88 27.22 36.67 36.02 35.99 37.80 Mean 24.85 30.02 34.30 37.32 27.24 27.47 28.89 27.27 26.92 27.54 36.12 37.08 35.68 36.47 cp o w Pooled SEM = 0.57 .6.
LSD = 1.61 o o c,.) Phosphorus Equivalency Estimates KH2PO4Standard Curve: Y = tibia ash (mg) o X = supplemental or equivalent P intake (g) =
w Y= 187.9 +393.4X
r2 = 0.95 For 500 U/kg Phytase activity (example calculations using tibia ash treatment means) %
Bioavailable P
Natuphos 1: (224 - 187.9)/393.4 = 0.092 g P from 351 g FT = 0.026%
r) Natuphos 2: (236 - 187.9)/393.4 = 0.122 g P from 373 g FT = 0.033%

I.) Ronozyme 1: (227 - 187.9)/393.4 = 0.099 g P from 349 g FT = 0.028%
a, (5) in Ronozymeg 2: (223 - 187.9)/393.4 = 0.089 g P from 326 g FT = 0.027%
I.) Mutant U: (404 - 187.9)/393.4 = 0.549 g P from 429 g FT = 0.128%
4. "
AppA: (403 - 187.9)/393.4 = 0.547 g P from 444 g FT = 0.123%

AppA2: (385 - 187.9)/393.4 = 0.501 g P from 437 g FI = 0.115%
a, AppA2/p: (409 - 187.9)/393.4 = 0.562 g P from 449 g FT = 0.125%

a, Results from ANOVA (calculation performed for each pen of four birds;
treatment legend on previous page) K) Bioavailable P (/o) 6 7 8 9 10 11 12 . 13 R1 0.048 0.028 0.042 0.036 0.058 0.029 0.133 0.117 0.123 0.138 R2 0.008 0.008 0.038 0.038 0.009 0.042 0.133 0.137 0.101 0.090 , R3 0.035 0.047 0.059 0.032 0.029 0.030 0.129 0.101 0.116 0.143 1-d R4 0.022 0.051 0.059 0.024 0.017 0.040 0.129 0.144 0.111 0.120 n ,-i R5 0.017 0.028 0.043 0.011 0.025 0.048 0.117 0.116 0.123 0.139 cp Mean 0.026' 0.032bc 0.048b 0.028` 0.028c 0.038be 0.128a - 0.123' 0.115' 0.125' =
w Pooled SEM = 0.006 .6.
vD
LSD = 0.018 o . c,.) At 500 U/kg, the yeast-expressed enzymes (Mutant U, AppA, AppA2, and AppA2/p) were more effective than Natuphos or Ronozyme at improving the in vivo responses tested including weight gain, feed to weight gain ratio, bone mass and mineral content, and percent bioavailable phosphate. The yeast-expressed enzymes were about four times more effective at increasing the level of bioavailable phosphate than either of the fungus-expressed enzymes.

IN VIVO EFFECTS OF YEAST-EXPRESSED
PHYTASES FED TO POST-MOLT LAYING HENS
The procedure was as described in Example 8 except post-molt laying hens were tested, egg production and egg weight was determined, and the treatment groups and basal diet were as follows:
Treatments:
1. P-deficient corn-soybean meal basal diet (0.10% Pa; 3.8% Ca; 17% CP) 2. As 1 + 0.10% Pi from KH2PO4 3. As 1 + 150 U/kg r-AppA2 phytase 4. As 1 + 300 U/kg r-AppA2 phytase 5. As 1 + 10,000 U/kg r-AppA2 phytase Basal Diet:
Ingredient Corn 63.65 Soybean meal, dehulled 25.65 Limestone, ground 9.80 Salt 0.40 Mineral premix 0.20 Vitamin premix 0.15 DL-methionine, feed-grade 0.10 Choline chloride 0.05 **Note: Treatment 1 discontinued after week 4 due to egg production below 50%.
The following tables are labeled as described in Example 8 and some of the same responses as described in Example 8 were measured. The results show that AppA2 increases egg production and egg weight in post-molt laying hens.

Treatments:
1. P-deficient corn-soybean meal basal diet (0.10% pa; 3.8% Ca; 17%
CP) 2. As 1 + 0.10% Pi from K.H2PO4 3. As 1 + 150 U/kg r-AppA2 phytase 4. As 1 + 300 U/kg r-AppA2 phytase 5. As 1 + 10,000 U/kg r-AppA2 phytase Initial body weights (g; mean of 12 hens) Ti T2 T3 T4 T5 mean 1716 1725 1733 1798 1746 Pooled SEM = 26 LSD =78 4-wk body weights (g: mean of 12 hens) Ti T2 T3 T4 T5 mean 1593 1748 1771 1806 1770 Pooled SEM = 21 LSD =64 12-wk body weights (g: mean of 12 hens) Ti T2 T3 T4 T5 R1 -- 1876 , 1831 1792 , 1781 , R2 -- 1791 1775 1856 1791 R3 -- 1800 . 1765 1806 1933 . R4 -- 1853 1814 1876 1815 mean -- 1830 1796 1833 1830 Pooled SEM = 24 LSD = 74 Treatments:
1. P-deficient corn-soybean meal basal diet (0.10% pa; 3.8% Ca; 17%
CP) 2. As 1 + 0.10% Pi from KH2PO4 3. As 1 + 150 U/kg r-AppA2 phytase 4. As 1 + 300U/kg r-AppA2 phytase 5. As 1 + 10,000 U/kg r-AppA2 phytase Feed intake(g/h/d)1 Ti T2 T3 T4 T5 mean 91b 12a 118a 117a 121a Pooled SEM =2 LSD = 5 Means are average daily feed intakes of hens for the first 4-wk period for treatment 1, and for the entire 12-wk period for treatments 2-5.
Egg weights (g)1 Ti T2 T3 T4 T5 R1 57.5 64.0 65.4 65.7 64.5 R2 63.5 64.7 64.3 66.0 65.5 R3 60.3 64.3 64.6 64.8 65.6 R4 62.8 63.3 62.2 65.3 63.7 mean 61.0b 64.1a 64.1a 65.5a 64.8a Pooled SEM = 0.7 LSD = 2.2 1 Means are average egg weights of hens for the first 4-wk period for treatment 1, and for the entire 12-wk period for treatments 2-5.

.
=
7:-:--, -.1 Treatments:
o i..) 1. P-deficient corn-soybean meal basal diet (0.10% pa; 3.8% Ca; 17% CP) 2. As 1 + 0.10% Pi from KH2PO4 3. As 1 + 150 U/kg r-AppA2 phytase 4. As 1 + 300 U/kg r-AppA2 phytase 5. As 1 + 10,000 U/kg r-AppA2 phytase Egg production by week (YO) n 1 2 3 4 5 6 7 8 9 = 10 D1 75.3 55.7 36.0 22.9 -- -- -- -- -- --I.) a, D2 88.4 90.8 88.1 87.8 88.4 85.4 86.0 81.8 80.4 79.8 80.7 78.3 0, u-, D3 84.5 85.1 83.3 85.1 83.3 82.1 83.6 79.2 77.4 77.4 79.5 76.5 I.) D4 86.6 86.3 83.9 82.4 82.1 84.5 81.5 77.4 78.0 74.7 73.8 72.0 D5 82.4 83.3 83.6 84.8 80.7 81.3 82.7 78.6 80.1 78.9 76.8 72.6 ...
a, SEM 3.0 3.2 3.4 3.5 3.3 3.2 3.2 4.1 3.4 4.5 3.5 3.9 0 a, I.) Egg production ( /0)1 co Ti T2 T3 T4 R1 44.6 86.5 73.0 81.0 80.7 R2 60.1 85.7 78.1 81.7 74.7 R3 43.2 87.2 84.3 83.9 87.8 R4 42.0 80.9 90.8 74.6 85.3 mean 47.5 85.1 81.6 80.3 82.1 1-d Least-squares means2 53.8b . 81.2a 80.7a 77.8' 82.9a n ,-i Pooled SEM = 2.1 cp Means are the average egg production of hens for the first 4-wk period for treatment 1, and for the o t..) entire 12-wk period for treatments 2-5.

.6.
2 Due to variation in week 1 egg production (above), covariance was used to analyze overall egg o o production, with least-squares means showing the effect of the covariable.
, IN VIVO EFFECTS OF YEAST-EXPRESSED
PHYTASES FED TO FINISHING PIGS
The procedure was as described in Example 8 except finishing pigs (i.e., gilts and barrows) were tested and the basal diet and treatment groups were as follows:
Treatments:
1. P-deficient corn-soybean meal basal diet 2. As 1 + 0.10% Pi from KH2PO4 3. As 1 + 250 FTU/kg r-AppA2 phytase 4. As 1 + 500 FTU/kg r-AppA2 phytase 5. As 1 + 1,000 FTU/kg r-AppA2 phytase 6. As 1 + 10,000 FTU/kg r-AppA2 phytase Basal diets:
Weight range (kg) Ingredient 50-80 80-120 Cornstarch to 100 to 100 Corn 78.42 83.85 Soybean meal, dehulled 18.08 12.65 Limestone, ground 1.06 1.07 Dicalcium phosphate 0.16 Trace mineral premix 0.35 0.35 Vitamin premix 0.10 0.10 L-Lysine-HC1, feed-grade 0.16 0.11 L-threonine, feed-grade 0.02 Antibiotic premix 0.75 0.75 Calculated composition (NRC, 1998) Crude protein, % 15.1 13.0 Lysine, total % 0.88 0.69 Calcium, % 0.50 0.45 Phosphorus, total % 0.38 0.32 Phosphorus, estimated bioavailable, % 0.09 0.05 ME, kcal/kg 3293 3295 The following tables are labeled as described in Example 8 and some of the responses described in Example 8 were measured. Gain/feed ratio is shown rather than a feed/gain ratio.
The results show that AppA2 was as effective as phosphate at increasing bone mass and mineral content, and at improving the gain/feed ratio.

Treatments:
1. P-deficient corn-soybean meal basal diet 2. As 1 + 0.10% Pi from KH2PO4 3. As 1 + 250 FTU/kg r-AppA2 phytase 4. As 1 + 500 FTU/kg r-AppA2 phytase 5. As 1 + 1,000 FTU/kg r-AppA2 phytase 6. As 1 + 10,000 FTU/kg r-AppA2 phytase Initial pig weights (kg) Barrows Gilts R1 52.2 , 52.8 53.0 51.8 52.0 51.8 51.2 50.8 , 52.2 52.2 51.3 52.4 R2 51.0 51.1 51.7 50.3 51.6 50.4 49.6 49.6 50.3 50.0 50.4 50.8 R3 48.2 49.6 49.8 49.6 50.1 , 49.2 48.1 49.6 47.9 , 47.1 48.8 48.4 R4 46.4 46.5 46.5 46.9 47.4 47.9 45.9 45.4 44.3 46.6 46.5 45.7 R5 52.0 44.1 51.0 52.4 46.4 50.7 43.4 43.9 44.0 43.1 44.1 44.0 mean 50.0 48.8 50.4 50.2 49.5 50.0 47.6 47.9 47.7 47.8 48.2 48.3 Pooled SEM = 0.4 Phase-switch pig weights (kg) Barrows Gilts R1 78.8 83.0 85.0 76.0 79.6 79.2 79.7 80.1 88.6 84.1 83.1 89.6 R2 76.8 80.6 86.9 79.9 82.2 83.8 80.0 83.5 87.7 84.5 87.3 83.5 R3 73.7 79.8 77.1 79.1 79.0 75.9 77.1 77.6 81.3 79.9 82.6 82.4 R4 82.3 82.5 79.2 79.1 84.4 84.5 74.7 78.1 73.9 84.6 78.9 79.1 R5 84.5 78.5 84.7 83.2 85.3 85.2 _ 83.3 80.5 84.4 87.2 81.7 82.5 mean 79.2 80.9 82.6 79.5 82.1 81.7 78.9 79.9 83.2 83.4 82.7 83.4 Pooled SEM = 0.8 Final pig weights (kg) Barrows Gilts R1 111.3 121.2 121.9 115.9 112.9 111.1 105.9 109.5 119.9 116.1 105.4 130.1 R2 111.5 119.6 132.7 111.9 121.3 116.3 105.8 115.6 118.3 115.3 123.3 112.5 R3 115.9 126.4 117.1 119.9 114.0 120.7 104.9 107.9 123.6 , 125.2 127.1 130.8 R4 116.6 117.9 110.0 110.0 119.7 122.1 120.2 121.6 117.9 127.7 109.3 123.0 R5 118.3 111.6 122.3 114.2 123.0 117.1 115.2 110.4 119.2 135.1 119.1 117.1 mean 114.7 119.3 120.8 114.4 118.2 117.5 110.3 113.0 119.8 123.9 116.8 122.7 Pooled SEM = 3.0 (Sex x Diet, P<0.10) Contrasts: Sex x Pi (2) vs Phytase (3-6), P<0.05.

Treatments:
1. P-deficient corn-soybean meal basal diet 2. As 1 + 0.10% Pi from KH2PO4 3. As 1 + 250 FTU/kg r-AppA2 phytase 4. As 1 + 500 FTU/kg r-AppA2 phytase 5. As 1 + 1,000 FTU/kg r-AppA2 phytase 6. As 1 + 10,000 FTU/kg r-AppA2 phytase Weight gain, initial-switch (g/d) Barrows Gilts R2 993 1135 1353 . 1140 1178 1283 867 966 1068 986 1055 936 R3 979 1162 1048 . 1131 1109 1028 829 801 955 935 965 mean 990 1094 1107 1001 1103 1079 857 882 974 976 950 968 Pooled SEM = 0.24 Contrasts: Barrows (Ba) vs Gilts (Gi), P<0.01; 1 vs 2-6, P<0.01 Weight gain, switch-final(g/d) Barrows Gilts R2 890 1000 1174 822 1001 833 . 688 874 835 830 974 784 R5 912 895 1018 836 1020 864 531 499 581 798 .

mean 896 972 968 881 918 901 673 723 790 857 734 853 Pooled SEM = 37 Contrasts: Ba vs Gi, P< 0.05 Weight gain, overall (g/d) Barrows Gilts R1 909 1053 1060 986 937 913 760 814 940 887 752 . 1079 R4 975 1006 882 876 1004 1030 864 887 855 . 944 730 898 R5 922 938 992 858 1063 922 703 652 738 901 . 735 717 mean 935 1023 1023 929 993 974 752 790 872 909 828 902 Pooled SEM = 38 (Sex x diet, P<0.10) Contrasts: Sex x Pi (2) vs Phytase (3-6), P<0.05 Treatments:
1. P-deficient corn-soybean meal basal diet 2. As 1 + 0.10% Pi from KH2PO4 3. As 1 + 250 FTU/kg r-AppA2 phytase 4. As 1 + 500 FTU/kg r-AppA2 phytase 5. As 1 + 1,000 FTU/kg r-AppA2 phytase 6. As 1 + 10,000 FTU/kg r-AppA2 phytase Feed intake, initial-switch (g/d) Barrows Gilts mean 2505 2496 2628 2352 2551 2371 2165 1927 2337 2189 2107 2146 Contrasts: Ba vs Gi, P<0.01 Feed intake, switch-final (g/d) Barrows Gilts R1 3181 3427 3559 3270 2962 2918_ 2443 2615 2890 2651 2094 3739 R2 2922 3039 3833 3011 3141 3147 2481 2652 2936 . 2796 3316 2565 R4 2978 . 2945 2872 2646 3104 2876 2935 2946 , 2373 2685 2481 2836 mean 3082 3140 3332 2982 3126 2926 2482 2414 2755 2754 2587 2895 Pooled SEM = 105 Feed intake, overall (g/d) Barrows Gilts 2632 2599_ 2432 , 2790 2488 2597 2655 2188 2463 . 2391 2583 mean 2837 2861 3028 2712 2873 2684 2347 2197 2571 2507 2378 2562 Pooled SEM =81 Treatments:
1. P-deficient corn-soybean meal basal diet 2. As 1 + 0.10% Pi from KH2PO4 3. As 1 + 250 FTU/kg r-AppA2 phytase 4. As 1 + 500 FTU/kg r-AppA2 phytase 5. As 1 + 1,000 FTU/kg r-AppA2 phytase 6. As 1 + 10,000 FTU/kg r-AppA2 phytase Gain/feed, initial-switch (g/kg) Barrows Gilts mean 397 438 420 425 433 458 397 474 418 446 461 454 Pooled SEM = 12 Contrasts: 1 vs. 2-6, P<0.01; 3 vs 4-6, P<0.10 Gain/feed, switch-final (g/d) Barrows Gilts mean 291 310 291 296 293 310 269 298 289 312 283 293 Pooled SEM =8 Gain/feed overall (g/d) Barrows Gilts mean 331 358 338 343 346 365 320 363 341 363 349 352 Pooled SEM =8 Contrasts: 1 vs 2-6, P<0.01 Treatments:
1. P-deficient corn-soybean meal basal diet 2. As 1 + 0.10% Pi from KH2PO4 3. As 1 + 250 FTU/kg r-AppA2 phytase 4. As 1 + 500 FTU/kg r-AppA2 phytase 5. As 1 + 1,000 FTU/kg r-AppA2 phytase 6. As 1 + 10,000 FTU/kg r-AppA2 phytase Fibula Dry Weight (g) Barrows Gilts R1 8.18 10.90 11.45 12.11 . 10.08 12.08 7.54 10.95 9.92 9.42 9.87 11.29 R2 8.84 11.74 8.66 10.98 11.21 11.66 7.96 8.81 9.33 11.41 10.70 12.73 -R3 8.54 11.29 , 9.81 11.90 10.10 12.77 8.62 10.25 9.94 10.50 11.86 12.46 R4 9.82 10.69 9.06 10.22 11.05 12.40 8.26 , 11.61 , 9.67 10.92 10.91 10.49 R5 7.88 8.88 10.33 10.51 12.01 11.26 7.68 9.51 11.16 11.48 10.10 11.44 mean 8.65 10.70 9.86 11.14 10.89 12.03 8.01 10.23 10.00 10.75 10.69 11.68 Pooled SEM = 0.27 Contrasts: 1 vs 2-6, P<0.01; 3 vs 4-6, P<0.01 Fibula Ash Weight (g) Barrows Gilts R1 4.37 6.32 6.34 6.90 6.03 6.94 4.06 , 6.42 . 5.21 . 5.33 5.61 6.78 R2 4.26 7.05 5.12 , 6.49 6.24 , 6.80 4.36 5.18 5.51 6.25 6.21 7.16 R3 4.51 6.54 5.78 . 7.17 5.81 7.49 4.35 5.91 , 6.11 5.79 6.93 7.23 R4 5.34 6.35 . 5.19 5.90 6.73 7.33 , 4.28 7.13 . 5.66 6.35 6.56 6.22 R5 4.37 5.22 6.02 . 6.34 7.06 6.64 3.91 5.64 7.02 6.25 5.88 6.93 mean 4.57 6.30 5.69 6.56 6.37 7.04 4.19 6.06 5.90 5.99 6.24 6.86 Pooled SEM = 0.17 Contrasts: Ba vs Gi, P< 0.10; 1 vs 2-6, P<0.01; 3 vs 4-6, P<0.01; 4 vs 5-6, P<0.10;
5 vs 6, P<0.01 Fibula Ash Percent (%) Barrows Gilts R1 53.42 57.98 55.37 56.98 59.82 57.45 53.85 58.63 52.52 56.58 56.84 60.05 R2 48.19 60.05 59.12 59.11 55.66 58.32 54.77 58.80 59.06 54.78 58.04 .
56.25 R3 52.81 57.93 58.92 60.25 57.52 58.65 50.46 57.66 61.47 55.14 58.43 58.03 R4 54.38 59.40 57.28 57.73 60.90 59.11 51.82 61.41 58.53 58.15 60.13 59.29 R5 55.46 58.78 58.28 , 60.32 58.78 58.97 50.91 59.31 62.90 54.44 58.22 60.58 mean 52.85 58.83 57.79 58.88 58.54 58.50 52.36 59.16 58.90 55.82 58.33 58.84 Pooled SEM = 0.65 Contrasts: 1 vs 2-6, P<0.01 Treatments:
1. P-deficient corn-soybean meal basal diet 2. As 1 + 0.10% Pi from KH2PO4 3. As 1 + 250 FTU/kg r-AppA2 phytase 4. As 1 + 500 FTU/kg r-AppA2 phytase 5. As 1 + 1,000 FTU/kg r-AppA2 phytase 6. As 1 + 10,000 FTU/kg r-AppA2 phytase Metatarsal Dry Weight (g) Barrows Gifts R1 11.42 14.03 15.84 15.36 14.43 13.85 11.77 15.90 16.00 15.48 12.65 15.05 R2 11.89 14.52 13.27 14.26 . 13.73 15.06 11.66 13.74 14.14 14.19 13.75 14.87 R3 14.01 14.45 13.20 13.99 14.91 17.43 10.52 12.20 11.95 16.31 17.53 17.13 R4 12.25 14.38 12.54 15.99 15.26 17.01 11.68 13.49 13.16 14.20 12.77 14.23 R5 12.55 13.30 14.30 14.36 17.79 14.29 11.26 12.76 12.47 16.93 12.78 13.10 mean 12.42 14.14 13.83 14.79 15.22 15.53 11.38 13.62 13.54 15.42 13.90 14.88 Pooled SEM = 0.44 Contrasts: 1 vs 2-6, P<0.01; 3 vs 4-6, P<0.05 Metatarsal Ash Weight (g) Barrows Gilts R1 5.28 . 6.59 7.97 6.93 6.74 6.86 . 4.74 7.21 6.72 7.09 6.07 . 7.50 R2 6.81 7.10 5.94 6.74 6.32 7.44 4.84 6.28 6.40 6.55 6.71 7.07 R3 4.82 6.95 6.41 6.77 6.72 7.88 4.82 5.59 6.67 6.99 8.13 8.11 R4 4.83 6.81 . 6.26 7.73 7.88 7.48 . 4.86 7.27 5.92 7.15 6.97 7.13 R5 5.20 5.75 7.22 6.99 8.33 7.14 5.24 6.61 6.65 7.07 6.04 6.55 mean 5.39 6.64 6.76 7.03 7.20 7.36 4.90 6.59 6.47 6.97 6.78 7.27 Pooled SEM = 0.18 Contrasts: 1 vs 2-6, P<0.01; 3 vs 4-6, P<0.05 Metatarsal Ash Percent (%) Barrows Gilts R1 46.25 46.99 50.31 45.15 46.75 49.56 40.22 45.37 42.01 45.80 47.96 49.84 R2 39.90 48.90 44.75 . 47.16 . 46.02 49.39 41.50 45.72 45.27 46.18 48.80 47.55 R3 34.38 48.12 . 48.59 . 48.36 45.09 45.18 45.84 45.78 55.84 42.87 .
46.39 47.35 R4 39.44 47.27 49.89 48.36 . 51.65 43.98 41.63 53.93 44.97 50.32 54.59 50.11 R5 41.44 43.26 50.50 , 48.69 46.81 49.95 46.50 51.82 53.33 41.74 . 47.28 50.00 mean 40.28 46.91 48.81 47.54 47.26 47.61 43.14 48.52 48.28 45.38 49.00 48.97 Pooled SEM = 1.05 Contrasts: 1 vs 2-6, P<0.01 IN VIVO EFFECTS OF YEAST-EXPRESSED PHYTASES FED TO PIGS
The procedure was as described in Example 8 except that the treatment groups were as follows:
28-day period 1. Basal - .08% available phosphorus 2. Basal + .05 phosphorus from monosodium phosphate 3. Basal + .10 phosphorus from monosodium phosphate 4. Basal + .15 phosphorus from monosodium phosphate 5. Basal + 250 FTU/kg experimental phytase product 6. Basal + 500 FTU/kg experimental phytase product 7. Basal + 1,000 FTU/kg experimental phytase product 8. Basal + 2,000 FTU/kg experimental phytase product 9. Basal + Natuphos 500 FTU/kg The results are shown in the following table. The results show that AppA2 increases bone mass and mineral content and improves the gain/feed ratio as effectively as phosphate.

Effect of phytase supplementation on pig growth perfoimance and bone asha Added NaH2P0'1-120 0.00 0.05 0.10 0.15 Phytase units/d 250 500 1,000 2,000 500 S.E.
BASF
Daily gain, kg" 0.35g 0.391g 0.46de 0.49d 0.38g 0.42'1 0.47d 0.496 0.421 0.01 Daily feed intake, kg) 0.75f 0.75f 0.81def 0.85d 0.77ef 0.79clef 0.83def 0.85d 0.85de 0.07 G:Fuk 0.48f 0.53de 0.57d 0.58d 0.50ef 0.54de 0.57d 0.57d 0.49e1 0.02 0 Fibula ash, g" 0.57h 0.65gh 0.77f 0.88' 0.59' 0.72f 0.85' 0.97d 0.70fg 0.03 (5) Fibula ash, %" 34.6h 36.0gh 37.8g 41.5de 33.9h 38.2fg 40.4ef 42.6d 38.5f 0.84 % Available PI 18.43g 22.56g 38.31f 53.56' 66.71d 34.47f 4.07 aP Intake, g/d"I 0.58g 0.92g 1.45f 1.92' 0.68g 1.22f 1.81' 2.31d 1.17f 0.13 Supplemental aP Intake, 0.02' 0.34gh 0.84' 1.27' 0.12h 0.62fg 1.17e 1.64d 0.57fg 0.13 0 g/d'm 'Six replications of two pigs per pen for performance data; six replications of two pigs per pen for bone data except for the treatment with phytase added at 500 units/g, which has five replications.
Added P from monosodium phosphate (NaH2PO4F120) to the basal diet.
'Supplemental phytase added to the basal diet.
defghMeariS within a row without common superscripts differ (P <0.05).
'Linear effect of added P from monosodium phosphate (P <0.001).
-linear effect of supplemental phytase (P <0.001).
1-d kUF phytase v. BASF phytase (P <0.07) = n 'Assumes that the P in corn, soybean meal, and monosodium phosphate is 11.25, and 100% available, respectively.
'Assumes that the P in monosodium phosphate is 100% available.

IN VIVO EFFECTS OF YEAST-EXPRESSED
PHYTASES FED TO CHICKS AND PIGS
The procedure for the studies summarized in the following tables was as described in Example 8. The treatment groups are shown in each table and the basal diet compositions are shown in the following table (see next page). The results show that AppA2 (ECP) is as effective as phosphate in improving the gain/feed ratio and in increasing bone mass and mineral content. The results also show that AppA2 is more effective than Natuphos and Ronozyme0 at increasing bioavailable phosphate. Furthermore, the results show that AppA2 increases egg weight and egg production in laying hens as effectively as phosphate.

Percentage composition of diets (as-fed basis).
Finishing pig assay Chick Young pig 50-80 80-120 Laying Ingredient assays assay kg kg hen assay Cornstarch to 100 to 100 to 100 to 100 -Corn 50.89 60.85 78.42 83.85 63.65 Soybean meal, dehulled 39.69 31.19 18.08 12.65 25.65 Soybean oil 5.00 3.00 - - -Limestone, ground 1.67 1.06 1.06 1.07 9.80 Salt 0.40 - - - 0.40 Dicalcium phosphate - - 0.16 - -Trace mineral premix 0.15a035h 0.35h 0.35h 0.20a .
Vitamin premix 0.20' 0.20d 0.10d 0.10d 0.15' Choline Chloride (60%) 0.20 - - - 0.05 Antibiotic premix 0.05' 1.00f 0.75g 0.75g -Copper sulfate - 0.08 - - -L-Lysine HCI, feed grade - 0.17 0.16 0.11 -L-Threonine, feed grade - - 0.02 - -DL-Methionine, feed grade 0.20 0.05 - - 0.10 Chemical composition Crude protein, %h 22.6 20.8 15.1 13.0 17.0 Total phosphorus, %h 0.42 0.35 0.38 0.32 0.34 Available phosphorus, %i 0.10 0.075 0.09 0.05 0.07 Calcium, %I. 0.75 0.60 0.50 0.45 3.8 ME, kcal/ke 3123 3387 3293 3295 2758 aSupplied the following per kilogram of complete diet: Fe, 75mg (FeSO4H20);
Zn, 100mg (Zn0); Mn, 75mg (MnO); Cu, 8 mg (CuSO4H20); I, 0.35 mg (CaI2); Se, 0.3 mg (Na2Se03);
NaCL, 3 g.
hSupplied the following per kilogram of complete diet: Fe, 90 mg (FeSO4H20);
Zn, 100 mg (Zn0); Mn, 20 mg (MnO); Cu, 8 mg (CuS041120); I, 0.35 mg (CaI2); Se,Ø3 mg (Na2Se03), NaC1, 3 g.
'Supplied the following per kilogram of complete diet: retinyl acetate, 1,514 jug;
cholecalciferol, 25 lig; DL-a-tocopheryl acetate, 11 mg; menadione sodium bisulfite complex, 2.3 mg; niacin, 22 mg; D-Ca-pantothenate, 10 mg; riboflavin, 4.4 mg;
vitamin B12, 11 pg.
dSupplied the following per kilogram of complete diet; retinyl acetate, 2,273 jig;
cholecalciferol, 16.5 itg; DL-a-tocopheryl acetate, 88 mg; menadione, 4.4 mg (menadione sodium bisulfite complex); niacin, 33 mg; D-Ca-pantothenate, 24.2 mg;
riboflavin, 8.8 mg;
vitamin B12, 35 lig; choline chloride, 319 mg.
'Provided 50mg of bacitracin per kilogram of complete diet.
fProvided 55 mg of mecadox per kilogram of complete diet.
gProvided 38 mg of roxarsone per kilogram of complete diet.
hAnalyzed (AOAC, 1999).
'Calculated (NRC, 1994; NRC, 1998).

Assessment of relative phosphorus bioavailability in chicks as affected by two different phytase enzymes (Chick assay oa.
Weight Gain/feed, Tibia ash Bioavailable Diet gain, g g/kg mg P, %
1. Basal diet 259e 617d 28.1' 2641 2. As 1 + 0.05% Pi (KH2PO4) 290d 654c 32.2d 311e 3. As 1 + 0.10% P1(KH2PO4) 323c 639cd 36.4c 414d 4. As 1 + 500 FTU/kg Natuphose 289" 666c 30.0c 293' 0.027d 5. As 1 + 500 FTU/kg ECP 346c 656c 37.8c 448c 0.124c Pooled SEM 6 10 0.5 10 0.006 'Values are means of five pens of four male chicks fed the experimental diets during the period 8 to 22 d post-hatching; average initial weight was 91 g.
bThe linear regression of tibia ash (mg) for Diets 1 to 3 as a function of supplemental P intake (g) was Y = 257.1 9.8 + 299.0 30.7X (r2 = 0.88); Bioavailable P
concentrations (equivalent P yields) for Diets 4 and 5 were determined by calculating equivalent bioavailable P intake (g) from the standard curve, dividing that by the feed intake (g), and multiplying by 100.
c'd'e'fMeans within a column with different superscripts are different, P
<0.05.

Relative phosphorus bioavailability in chicks fed different phytase enzymes (Chick assay 2)a.
Weight Gain/feed, Tibia ash Bioavailable Diet gain, g g/kg % mg P, %
1. Basal diet 176k 569k 24.9k 183k -2. As 1 + 0.05% Pi (KH2PO4) 25311' 68011 30.0h .
27211 _ 3. As 1 +0.10% P1(KH2PO4) 293g 703Igh 34.3g 347g -4. As 1 +0.15% Pi (KH2PO4) 333t 73 1 et _ 37.3e 455e -. 5. As 1 + 500 FTU/kg Natuphosec 218 620 27.2? 224J
0.026g 6. As 1 + 500 FTU/kg Natuphosed 236" _ 632) 27.5" 236"
0.0321g 7. As 1 + 1,000 FTU/kg Natuphosed 265' 675gh 28.91"
26211' 0.048' 8. As 1 + 500 FTU/kg Ronozyme 2191 634J 26.9' 223i 0.028g 9. As 1 + 1,000 FTU/kg Ronozyme , 245' 682gh 27.5"
242h" 0.030 10. As 1 + 500 FTU/kg ECP 318e1 7081g11 36.5et 409' 0.125e , Pooled SEM 7 10 0.6 12 0.006 aValues are means of five pens of four male chicks fed the experimental diets during the period 8 to 22 d posthatching; average initial weight was 83 g.
hThe linear regression of tibia ash (mg) for Diets 1 to 4 as a function of supplemental P intake (g) was Y = 187.9 8.7 + 393.4 21.2X (r2 = 0.95); Bioavailable P
concentrations (equivalent P yields) for Diets 4-11 were determined by calculating equivalent bioavailable P
intake (g) from the standard curve, dividing that by the feed intake (g) of the pen, and multiplying by 100.
cEnzyme was from the same batch that was used for chick assay 1.
dEnzyme was from a different batch that was used for chick assay 1.
e'c'g'hij'kMeans within a column with different superscripts are different, P
<0.05.

The effect of activity level on the phosphorus-releasing efficacy of E. coli phytase in chicks (Chick assay 3)a.
Weight Gain/feed, Tibia ash Bioavailable Diet gain, g g/kg mg P, %b ¨
1. Basal diet _ 219h 661c 237' --2. As 1 + 0.05% Pi (KH2PO4) 283tg 69211 299h -3. As 1 + 0.10% Pi (KH2PO4) 314c 720c 413g -4. As 1 + 0.15% Pi (KH2PO4) 327dc 731c 490e -5. As 1 + 500 FTU/kg ECP 321dc 731c 447' 0.125c 6. As 1 + 1,000 FTU/kg ECP 335ed 732c 559d 0.183d 7. As 1 + 1,500 FTU/kg ECP , 344c 737c 616c 0.211c 8. As 1 + 500 FTU/kg Natuphos 276g 691d 290' 0.037' Pooled SEM 6 10 12 0.005 aValues are means of five pens of four male chicks fed the experimental diets during the period 8 to 22 d post-hatching; average initial weight was 97 g.
hThe linear regression of tibia ash (mg) for Diets 1 to 4 as a function of supplemental P intake (g) was Y = 232.0 6.9 + 389.9 16.7X (r2 = 0.97); Bioavailable P
concentrations (equivalent P yields) for Diets 5 to 8 were determined by calculating equivalent bioavailable P intake (g) from the standard curve, dividing that by the feed intake (g) of the pen, and multiplying by 100.
c,d,e,f'g'hAMeans within a column with different superscripts are different, P
<0.05.

Combining 3- and 6-phytases does not produce synergistic effects on Pi-release in chicks fed a corn-soybean meal diet(Chick assay 4)a.
Weight Gain/feed, Tibia ash Bioavailable Diet gain, g g/kg % mg P, %b 1. Basal diet 137g 610g 25.4g 134h -2. As 1 + 0.05% Pi (KH2PO4) 191d 678dc 29.01 1981g -3. As 1 + 0.10% Pi (KH2PO4) 225d 712d 32.8e 253e -4. As 1 + 0.15% Pi (KH2PO4) 276c _ 762c 36.3d 339d -5. As 1 + 500 FTU/kg Natuphos 192d 620 28.0' 187g 0.041g 6. As 1 + 500 FTU/kg Ronozyme 1821. 655et 27.7' 188g 0.0471g 7. As 1 + 500 FTU/kg ECP 272c 760c 37.0d 343d 0.153d 8. As 5 + 6 211de v 693de 28.31 2121g 0.064d 9. As 5 + 7 282c 763c 37.8d 360d 0.162' 10. As 1 + 1,000 FTU/kg Natuphos 217d _ 703d .
29.01 2171 0.067c 11. As 1 + 1,000 FTU/kg Ronozyme 201c1c1 . 666et 27.9' 1941g 0.050etg 12. As 1 + 1,000 FTU/kg ECP 292c 758c 41.1c 433c 0.206c Pooled SEM 9 15 0.6 10 0.007 aValues are means of five pens of four male chicks fed the experimental diets during the period 8 to 22 d post-hatching; average initial weight was 68 g.
hThe linear regression of tibia ash (mg) for Diets 1 to 4 as a function of supplemental P intake (g) was Y = 138.6 4.9 + 371.3 14.7X (r2 = 0.97); Bioavailable P
concentrations (equivalent P yields) for Diets 5 to 8 were determined by calculating equivalent bioavailable P intake (g) from the standard curve, dividing that by the feed intake (g) of the pen, and multiplying by 100.
c,d,e,fgMeans within a column with different superscripts are different, P
<0.05.

Effect of E. coli phytase on performance of laying hens from week 1-4.a Egg Egg Initial hen 4-wk hen Feed production, weight, Diet weight, g weight, g intake, g/d %b g 1. P-deficient basal diet 1716 1593 90 54.0 61.0 2. As 1+ 0.10% Pi 1725 1748 122 84.8 64.2 3. As 1 + 150 FTU/kg ECP 1733 1771 119 83.7 63.8 4. As 1 + 300 FTU/kg ECP 1798 1806 119 82.3 65.4 5. As 1 + 10,000 FTU/kg ECP 1746 1770 123 85.9 65.1 Pooled SEM 26 21' 2' 1.6' 0.7' aData are means of four replicates of 12 hens for the first 4 weeks of the study period.
bEgg production (%) analyzed using covariance; data presented are least-squares means.
'Diet 1 vs diets 2-5, P <0.01.
Effect of E. coli phytase on performance of laying hens from week 5-12a.
Egg Egg 4-wk hen Feed production, weight, Diet weight, g intake, g/d %b g 2. As 1+ 0.10% Pi 1830 120 80.5 64.0 3. As 1 + 150 FTU/kg ECP 1796 118 80.6 64.1 4. As 1 + 300 FTU/kg ECP 1833 116 77.2 65.5 5. As 1 + 10,000 FTU/kg ECP 1830 120 81.2 64.8 Pooled SEM 24 2 2.5 0.5' aData are means of four replicates of 12 hens for weeks 5 through 12 of the study period.
Diet 1 was removed from study due to poor egg production.
bEgg production (%) analyzed using covariance; data presented are least-squares means.
'Diet 3 vs diets 4 and 5, P <0.01.
Effect of E. coli phytase on performance of laying hens from week 1-12a.
Hen weights Feed Egg Egg intake, production, weight, Diet , Initial 4-wk 12-wk g/d %b g 1. P-deficient basal diet 1716 1593 - 90 53.8 .
61.0 2. As 1 + 0.10% PI . 1725 _ 1748 1830 121 81.2 _ 64.1 3. As 1 + 150 FTU/kg ECP 1733 1771 1796 _ 118 80.7 64.1 4. As 1 + 300 FTU/kg ECP 1798 _ 1806 1833 117 77.8 65.5 5. As 1 + 10,000 FTU/kg ECP 1746 _ 1770 1830 121 82.9 64.8 . .
Pooled SEM 26 21c 24 2' 2.1' 0.7' aData are means of four replicates of 12 hens. Data are means for the first 4 weeks for diet 1, but for all 12 weeks for diets 2-5.
bEgg production (%) analyzed using covariance; data presented are least-squares means.
'Diet 1 vs diets 2-5, P <0.01.

Relative bioavailability of phosphorus in young pigs fed different phrase enzymes (Pig assay 1).
Weight Gain/feed, Fibula ash B
ioavailable Diet gain, g/da g/kga mg P, 0/0C
1. Basal diet 369' 533' 29.3g 666' 2. As 1 + 0.05% Pi (KH2PO4) 435e 576 32.8' 766hl 3. As 1 + 0.10% Pi (KH2PO4) 476e 618e 36.6d 972e1 4. As 1 + 0.15% Pi (KH2PO4) 509" 660" 36.6" 1123"
5. As 1 + 400 FTU/kg Natuphos 460e 605e 34.4de1 880 0.081de 6. As 1 + 400 FTU/kg Ronozyme 445e 565e' 33.5 805gh 0.043' 7. As 1 + 400 FTU/kg ECP 443e 583e1 35.0det 968et 0.108"
Pooled SEM 17 21 0.8 38 0.016 aData are means of 10 individually-fed pigs over a 23-d feeding period;
average initial weight was 8.4 kg.
bData are means of five individually-fed pigs, chosen from the median-weight blocks at the end of the 23-d feeding period.
eThe linear regression of fibula ash (mg) for Diets 1 to 4 as a function of supplemental P
intake (g) was Y = 664.5 25.5 + 15.3 1.4X (r2 = 0.87); Bioavailable P
concentrations (equivalent P yields) for Diets 5-7 were determined by calculating equivalent bioavailable P
intake (g) from the standard curve, dividing that by the feed intake (g) of the pig, and multiplying by 100.
d,e,f,g,h,imeans within a column with different superscripts are different, P <0.05.

=
-a-, Effect of E. coil phytase on growth performance of finishing pigs (Pig assay 2)a. =
t..) Dietary treatment P-deficient As 1 + As 1 + 250 As 1 + 500 As 1 + 1,000 As 1 + 10,000 Pooled Response variable basal diet 0.10% Pi FTU/kg ECP FTU/kg ECP FTU/kg ECP FTU/kg ECP SEM
Daily gain, gb Barrows 935 1023 1023 929 Gilts 752 790 872 909 Mean 844 . 907 947 919 I.) a, Daily feed, g' (5) in I.) Barrows 2837 2861 3028 2712 .
I.) Gilts 2347 2197 2571 2507 2378 2562 0, i a, Mean 2592 2529 2800 2610 a, I.) Gain/fed, g/kgd Barrows 331 358 338 343 Gilts 320 363 341 363 Mean 325 361 339 353 'Data are means of five individually-fed pigs of each sex fed their experimental diets from 48.9 to 117.6 kg body weight.
bSex x diet interaction. P <0.10; Sex x Pi vs phytase-supplemented dies, P
<0.05.
1-d 'Barrows vs gilts, P <0.01.
n dP-deficient vs Pi- and phytase-supplemented diets, P <0.01.
cp o t..) .6.
o o c,.) =
=

o 'a Effect of E. coil phytase on bone characteristics of finishing pigs (Pig assay 2)d. --4 1--, o Dietary treatment n.) P-deficient As 1 + As 1 + 250 As 1 + 500 As 1 + 1,000 As 1 + 10,000 Pooled Response variable basal diet 0.10% Pi F1U/kg ECP FTU/kg ECP
FTU/kg ECP FTU/kg ECP SEM
Fibula ash, %gb Barrows 52.9 58.8 57.8 58.9 58.5 58.5 Gilts 52.4 59.2 58.9 55.8 58.3 58.8 Mean 52.6 59.0 58.3 57.3 58.4 58.7 0.7 n Fibula ash, gbcdef N
Barrows 4.57 6.30 5.69 6.56 6.37 7.04 a, c7, Gilts 4.19 6.06 5.90 5.99 6.24 6.86 in I.) .

0, I.) Mean 4.38 6.18 5.80 6.28 6.31 6.95 0.17 ---1 i I.) a, Metatarsal ash, %b Barrows 40.3 46.9 48.8 47.5 47.3 47.6 a, Gilts 43.1 . 48.5 48.3 45.4 49.0 49.0 I.) Mean 41.7 47.7 48.5 46.5 48.1 48.3 1.1 -Metatarsal ash, gbd Barrows 5.4 6.6 6.8 7.0 7.2 7.4 Gilts 4.9 6.6 6.5 7.0 6.8 7.3 Iv Mean . 5.1 6.6 6.6 7.0 7.0 7.3 0.2 n 'Data are means of five individually-fed pigs of each sex fed their experimental diets from 48.9 to 117.6 kg body weight.
bP-deficient vs Pi- and phytase-supplemented diets, P<0.01.
cp o 'Barrows vs gilts, P <0.10.
n.) c.:.) d250 U/kg vs higher phytase activity levels, P<0.01.
.6.
o '500 U/kg vs 1,000 and 10,000 U/kg phytase, P<0.10.
o f1,000 U/kg vs 10,000 U/kg phytase, P<0.01.

834460-71726.txt SEQUENCE LISTING
<110> Phytex, L.L.C.
Webel, Douglas M.
Orr, Donald E.
Ruch, Frank E.
<120> PHYTASE-CONTAINING ANIMAL FOOD AND METHOD
<130> 834460-71726 <150> US 60/335,303 <151> 2001-10-31 <160> 14 <170> PatentIn version 3.1 <210> 1 <211> 1489 <212> DNA
<213> Escherichia coil <220>
<221> primer_bind <222> (1)..(22) <223>
<220>
<221> primer_bind <222> (1468)..(1489) <223>

834460-71726.txt <220>
<221> cos <222> (16)..(108) <223>
<220>
<221> CDS
<222> (182)..(1480) <223>
<400> 1 taaggagcag aaaca atg tgg tat ttc ctt tgg ttc gtc ggc att ttg ttg 51 Met Trp Tyr Phe Leu Trp Phe Val Gly Ile Leu Leu atg tgt tcg ctc tcc acc ctt gtg ttg gta tgg ctg gac ccg cga ttg 99 met Cys Ser Leu Ser Thr Leu Val Leu val Trp Leu Asp Pro Arg Leu aaa agt taa cgaacgtaag cctgatccgg cgcattagcg tcgatcaggc 148 Lys Ser aataatatcg gatatcaaag cggaaacata tcg atg aaa gcg atc tta atc cca 202 Met Lys Ala Ile Leu Ile Pro ttt tta tct ctt ttg att ccg tta acc ccg caa tct gca ttc gct cag 250 Phe Leu Ser Leu Leu Ile Pro Leu Thr Pro Gin Ser Ala Phe Ala Gin agt gag ccg gag ctg aag ctg gaa agt gtg gtg att gtc agc cgt cat 298 Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg His ggt gtg cgt gcc cca acc aag gcc acg caa ctg atg cag gat gtc acc 346 Gly Val Arg Ala Pro Thr Lys Ala Thr Gin Leu Met Gin Asp val Thr cca gac gca tgg cca acc tgg ccg gta aaa ctg ggt tgg ctg aca cca 394 Pro Asp Ala Trp Pro Thr Trp Pro val Lys Leu Gly Trp Leu Thr Pro cgc ggt ggt gag cta atc gcc tat ctc gga cat tac caa cgc cag cgt 442 Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Gin Arg Gin Arg ctg gtg gcc gac gga ttg ctg gcg aaa aag ggc tgc ccg cag cct ggt 490 Leu val Ala Asp Gly Leu Leu Ala Lys Lys Gly Cys Pro Gin Pro Gly 834460-71726.txt cag gtc gcg att att gct gat gtc gac gag cgt acc cgt aaa aca ggc 538 Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr Gly gaa gcc ttc gcc gcc ggg ctg gca cct gac tgt gca ata acc gta cat 586 Glu Ala Phe Ala Ala Gly Leu Ala Pro AS Cys Ala Ile Thr Val His acc cag gca gat acg tcc agt ccc gat ccg tta ttt aat cct cta aaa 634 Thr Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu Lys act ggc gtt tgc caa ctg gat aac gcg aac gtg act gac gcg atc ctc 682 Thr Gly Val Cys Gln Leu Asp Asn Ala Asn Val Thr Asp Ala Ile Leu agc agg gca gga ggg tca att gct gac ttt acc ggg cat cgg caa acg 730 Ser Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Arg Gln Thr gcg ttt cgc gaa ctg gaa cgg gtg ctt aat ttt tcc caa tta aac ttg 778 Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Ser Gln Leu Asn Leu tgc ctt aac cgt gag aaa cag gac gaa agc tgt tca tta acg cag gca 826 Cys Leu Asn Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln Ala tta cca tcg gaa ctc aag gtg agc gcc gac aat gtt tca tta acc ggt 874 Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr Gly gcg gta agc ctc gca tca atg ctg acg gaa ata ttt ctc ctg caa caa 922 Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln Gln gca cag gga atg ccg gag ccg ggg tgg gga agg atc act gat tca cac 970 Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser His cag tgg aac acc ttg cta agt ttg cat aac gcg caa ttt tat tta cta 1018 Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Tyr Leu Leu caa cgc acg cca gag gtt gcc cgc agt cgc gcc acc ccg tta ttg gat 1066 Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu Asp ttg atc atg gca gcg ttg acg ccc cat cca ccg caa aaa cag gcg tat 1114 Leu Ile Met Ala Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala Tyr ggt gtg aca tta ccc act tca gtg ctg ttt att gcc gga cac gat act 1162 Gly val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp Thr 345 350 355 .
aat ctg gca aat ctc ggc ggc gca ctg gag ctc aac tgg acg ctt cca 1210 Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu Pro ggt cag ccg gat aac acg ccg cca ggt ggt gaa ctg gtg ttt gaa cgc 1258 Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu Arg 834460-71726.txt tgg cgt cgg cta agc gat aac agc cag tgg att cag gtt tcg ctg gtc 1306 Trp Arg Arg Leu Ser Asp Asn Ser Gin Trp Ile Gin Val Ser Leu Val ttc cag act tta cag cag atg cgt gat aaa acg ccg cta tca tta aat 1354 Phe Gin Thr Leu Gin Gin Met Arg Asp Lys Thr Pro Leu Ser Leu Asn acg ccg ccc gga gag gtg aaa ctg acc ctg gca gga tgt gaa gag cga 1402 Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu Arg aat gcg cag ggc atg tgt tcg ttg gcc ggt ttt acg caa atc gtg aat 1450 Asn Ala Gin Gly Met Cys Ser Leu Ala Gly Phe Thr Gin Ile Val Asn gaa gcg cgc ata ccg gcg tgc agt ttg taa tggtacccc 1489 Glu Ala Arg Ile Pro Ala Cys Ser Leu <210> 2 <211> 30 <212> PRT
<213> Escherichia coli <400> 2 Met Trp Tyr Phe Leu Trp Phe Val Gly Ile Leu Leu Met Cys Ser Leu Ser Thr Leu Val Leu Val Trp Leu Asp Pro Arg Leu Lys Ser <210> 3 <211> 432 <212> PRT
<213> Escherichia coli <400> 3 Met Lys Ala Ile Leu Ile Pro Phe Leu Ser Leu Leu Ile Pro Leu Thr Pro Gin Ser Ala Phe Ala Gin Ser Glu Pro Glu Leu Lys Leu Glu Ser 834460-71726.txt Val Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr Gin Leu Met Gin Asp val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Trp Leu Thr Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Gin Arg Gin Arg Leu Val Ala Asp Gly Leu Leu Ala Lys Lys Gly Cys Pro Gin Pro Gly Gin Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val His Thr Gin Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu Lys Thr Gly Val Cys Gin Leu Asp Asn Ala Asn Val Thr Asp Ala Ile Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Arg Gin Thr Ala Phe Arg Glu Leu Glu Arg val Leu Asn Phe Ser Gin Leu Asn Leu Cys Leu Asn Arg Glu Lys Gin Asp Glu Ser Cys Ser Leu Thr Gin Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gin Gin Ala Gin Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser His Gin Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gin Phe Tyr Leu Leu Gin Arg Thr Pro Glu Val Ala Arg Ser 834460-71726.txt Arg Ala Thr Pro Leu Leu Asp Leu Ile Met Ala Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln val Ser Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu val Lys Leu Thr Leu Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu <210> 4 <211> 1486 <212> DNA
<213> Escherichia coli <220>
<221> CDS
<222> (188)..(1483) <223>
<400> 4 taaggagcag aaacaatgtg gtatttactt tggttcgtcg gcattttgtt gatgtgttcg 60 ctctccaccc ttgtgttggt atggctggac ccgcgattga aaagttaacg aacgtaggcc 120 tgatgcggcg cattagcatc gcatcaggca atcaataatg tcagatatga aaagcggaaa 180 834460-71726.txt catatcg atg aaa gcg atc tta atc cca ttt tta tct ctt ctg att ccg 229 Met Lys Ala Ile Leu Ile Pro Phe Leu Ser Leu Leu Ile Pro tta acc ccg caa tct gca ttc gct cag agt gag ccg gag ctg aag ctg 277 Leu Thr Pro Gin Ser Ala Phe Ala Gin Ser Glu Pro Glu Leu Lys Leu gaa agt gtg gtg att gtc agc cgt cat ggt gtg cgt gcc cca acc aag 325 Glu Ser Val Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys gcc acg caa ctg atg cag gat gtc acc cca gac gca tgg cca acc tgg 373 Ala Thr Gin Leu Met Gin Asp Val Thr Pro Asp Ala Trp Pro Thr Trp ccg gta aaa ctg ggt tgg ctg aca cca cgc ggt ggt gag cta atc gcc 421 Pro Val Lys Leu Gly Trp Leu Thr Pro Arg Gly Gly Glu Leu Ile Ala tat ctc gga cat tac caa cgc cag cgt ctg gtg gcc gac gga ttg ctg 469 Tyr Leu Gly His Tyr Gin Arg Gin Arg Leu Val Ala Asp Gly Leu Leu gcg aaa aag ggc tgc ccg cag cct ggt cag gtc gcg att att gtc gat 517 Ala Lys Lys Gly Cys Pro Gin Pro Gly Gin Val Ala Ile Ile val Asp gtc gac gag cgt acc cgt aaa aca ggc gaa gcc ttc gcc gcc ggg ctg 565 Val Asp Glu Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu gca cct gac tgt gca ata acc gta cat acc cag gca gat acg tcc agt 613 Ala Pro Asp Cys Ala Ile Thr Val His Thr Gin Ala Asp Thr Ser Ser ccc gat ccg tta ttt att cct cta aaa act ggc gtt tgc caa ctg gat 661 Pro Asp Pro Leu Phe Ile Pro Leu Lys Thr Gly Val Cys Gin Leu Asp aac gcg aac gtg act gac gcg atc ctc agc agg gca gga ggg tca att 709 Asn Ala Asn Val Thr Asp Ala Ile Leu Ser Arg Ala Gly Gly Ser Ile gct gac ttt acc ggg cat cgg caa acg gcg ttt cgc gaa ctg gaa cgg 757 Ala Asp Phe Thr Gly His Arg Gin Thr Ala Phe Arg Glu Leu Glu Arg gtg ctt aat ttt ccg caa tca aac ttg aac ctt aaa cgt gag aaa cag 805 Val Leu Asn Phe Pro Gin Ser Asn Leu Asn Leu Lys Arg Glu Lys Gin aat gaa agc tgt aac tta acg cag gca tta cca tcg gaa ctc aag gtg 853 Asn Glu Ser Cys Asn Leu Thr Gin Ala Leu Pro Ser Glu Leu Lys Val agc gcc gac aat gtt tca tta acc ggt gcg gta agc ctc gca tca atg 901 Ser Ala Asp Asn Val Ser Leu Thr Gly Ala val Ser Leu Ala Ser Met ctg acg gaa ata ttt ctc ctg caa caa gca cag gga atg ccg gag ccg 949 Leu Thr Glu Ile Phe Leu Leu Gin Gin Ala Gin Gly Met Pro Glu Pro 834460-71726.txt ggg tgg gga agg atc act gat tca cac cag tgg aac acc ttg cta agt 997 Gly Trp Gly Arg Ile Thr Asp Ser His Gln Trp Asn Thr Leu Leu Ser ttg cat aac gcg caa ttt tat tta cta caa cgc acg cca gag gtt gcc 1045 Leu His Asn Ala Gln Phe Tyr Leu Leu Gln Arg Thr Pro Glu val Ala cgc agt cgc gcc acc ccg tta ttg gat ttg atc aag aca gcg ttg acg 1093 Arg Ser Arg Ala Thr Pro Leu Leu Asp Leu Ile Lys Thr Ala Leu Thr ccc cat cca ccg caa aaa cag gcg tat ggt gtg aca tta ccc act tca 1141 Pro His Pro Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro Thr Ser gtg ctg ttt att gcc gga cac gat act aat ctg gca aat ctc ggc ggc 1189 Val Leu Phe Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly gca ctg gag ctc aac tgg acg ctt cca ggt cag ccg gat aac acg ccg 1237 Ala Leu Glu Leu Asn Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro cca ggt ggt gaa ctg gtg ttt gaa cgc tgg cgt cgg cta agc gat aac 1285 Pro Gly Gly Glu Leu Val Phe Glu Arg Trp Arg Arg Leu Ser Asp Asn agc cag tgg att cag gtt tcg ctg gtc ttc cag act tta cag cag atg 1333 Ser Gln Trp Ile Gln val Ser Leu val Phe Gln Thr Leu Gln Gln Met cgt gat aaa acg ccg cta tca tta aat acg ccg ccc gga gag gtg aaa 1381 Arg Asp Lys Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys ctg acc ctg gca gga tgt gaa gag cga aat gcg cag ggc atg tgt tcg 1429 Leu Thr Leu Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser ttg gcc ggt ttt acg caa atc gtg aat gaa gcg cgc ata ccg gcg tgc 1477 Leu Ala Gly Phe Thr Gln Ile val Asn Glu Ala Arg Ile Pro Ala Cys agt ttg taa 1486 Ser Leu <210> 5 <211> 432 <212> PRT
<213> Escherichia coil <400> 5 834460-71726.txt Met Lys Ala Ile Leu Ile Pro Phe Leu Ser Leu Leu Ile Pro Leu Thr Pro Gin Ser Ala Phe Ala Gin Ser Glu Pro Glu Leu Lys Leu Glu Ser val val Ile val Ser Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr Gin Leu met Gin Asp Val Thr Pro Asp Ala Trp Pro Thr Trp Pro val Lys Leu Gly Trp Leu Thr Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Gin Arg Gin Arg Leu Val Ala Asp Gly Leu Leu Ala Lys Lys Gly Cys Pro Gin Pro Gly Gin Val Ala Ile Ile Val Asp Val Asp Glu Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr val His Thr Gin Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Ile Pro Leu Lys Thr Gly Val Cys Gin Leu Asp Asn Ala Asn Val Thr Asp Ala Ile Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Arg Gin Thr Ala Phe Arg Glu Leu Glu Arg val Leu Asn Phe Pro Gin Ser Asn Leu Asn Leu Lys Arg Glu Lys Gin Asn Glu Ser Cys Asn Leu Thr Gin Ala Leu Pro Ser Glu Leu Lys val Ser Ala Asp Asn Val Ser Leu Thr Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln Gin Ala Gin Gly Met Pro Glu Pro Gly Trp 834460-71726.txt Gly Arg Ile Thr Asp Ser His Gin Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gin Phe Tyr Leu Leu Gin Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gin Lys Gin Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu Pro Gly Gin Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu Arg Trp Arg Arg Leu Ser Asp Asn Ser Gin Trp Ile Gin Val Ser Leu Val Phe Gin Thr Leu Gin Gin Met Arg Asp Lys Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gin Ile Val Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu <210> 6 <211> 80 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for amplifying appA gene.
<400> 6 ggggtaccat gggcgtctct gctgttctac ttcctttgta tctcctgtct ggagtcacct 60 ccggacagag tgagccggag 80 834460-71726.txt <210> 7 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for amplifying appA gene.
<400> 7 gggaattcat tacaaactgc aggc 24 <210> 8 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for amplifying appA gene.
<400> 8 ggaattccag agtgagccgg a 21 <210> 9 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for amplifying appA gene.
<400> 9 ggggtacctt acaaactgca cg 22 <210> 10 <211> 31 <212> DNA
<213> Artificial Sequence 834460-71726 . txt <220>
<223> Primer for amplifying appA mutagenesis PCR amplification.
<400> 10 ctgggtatgg ttggttatat tacagtcagg t 31 <210> 11 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for amplifying appA mutagenesis PCR amplification.
<400> 11 caaacttgaa ccttaaacgt gag 23 <210> 12 <211> 31 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for amplifying appA mutagenesis PCR amplification.
<400> 12 cctgcgttaa gttacagctt tcattctgtt t 31 <210> 13 <211> 30 <212> PRT
<213> Escherichia coil <400> 13 Met Trp Tyr Phe Leu Trp Phe Val Gly Ile Leu Leu Met Cys Ser Leu 834460-71726.txt Ser Thr Leu Val Leu Val Trp Leu Asp Pro Arg Leu Lys Ser <210> 14 <211> 432 <212> PRT
<213> Escherichia coil <400> 14 Met Lys Ala Ile Leu Ile Pro Phe Leu Ser Leu Leu Ile Pro Leu Thr Pro Gin Ser Ala Phe Ala Gin Ser Glu Pro Glu Leu Lys Leu Glu Ser val val Ile val Ser Arg His Gly val Arg Ala Pro Thr Lys Ala Thr Gin Leu met Gin Asp val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Trp Leu Thr Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Gin Arg Gin Arg Leu Val Ala Asp Gly Leu Leu Ala Lys Lys Gly Cys Pro Gin Pro Gly Gin Val Ala Ile Ile Ala Asp val Asp Glu Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr val His Thr Gin Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu Lys Thr Gly val Cys Gin Leu Asp Asn Ala Asn Val Thr Asp Ala Ile Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Arg Gin Thr Ala Phe Arg Glu Leu Glu Arg Val Leu 834460-71726.txt Asn Phe Ser Gin Leu Asn Leu Cys Leu Asn Arg Glu Lys Gin Asp Glu Ser Cys Ser Leu Thr Gin Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gin Gin Ala Gin Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser His Gin Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gin Phe Tyr Leu Leu Gin Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu Asp Leu Ile Met Ala Ala Leu Thr Pro His Pro Pro Gin Lys Gin Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu Pro Gly Gin Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu Arg Trp Arg Arg Leu Ser Asp Asn Ser Gin Trp Ile Gin Val Ser Leu Val Phe Gin Thr Leu Gin Gin Met Arg Asp Lys Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu Arg Asn Ala Gin Gly Met Cys Ser Leu Ala Gly Phe Thr Gin Ile Val Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu

Claims (71)

CLAIMS:

1. A method of improving the nutritional value of a foodstuff consumed by a monogastric animal wherein the foodstuff increases the bone mass and bone mineral content of the animal by increasing the bioavailability of phosphate from phytate wherein the foodstuff comprises myo-inositol hexakisphosphate, the method comprising the step of feeding to the animal the foodstuff in combination with less than 1200 units of an E. coli 6-phytase expressed in yeast per kilogram of the foodstuff and an encapsulating agent, wherein the bioavailability of phosphate from phytate is increased by at least 2-fold compared to the bioavailability of phosphate from phytate obtained by feeding the foodstuff in combination with the same units of a phytase expressed in a non-yeast host cell.

2. A method of improving the nutritional value of a foodstuff consumed by a monogastric animal wherein the foodstuff comprises myo-inositol hexakisphosphate, the method comprising the step of feeding to the animal the foodstuff in combination with an E. coli 6-phytase expressed in yeast and an encapsulating agent, wherein the bone mass and mineral content of the animal is increased.

3. A method of improving the nutritional value of a foodstuff consumed by a monogastric animal wherein the foodstuff increases the bone mass and bone mineral content of the animal, and wherein the foodstuff comprises myo-inositol hexakisphosphate, the method comprising the steps of:
spray drying an E. coli 6-phytase;
mixing the encapsulated phytase with a carrier for the phytase and, optionally, other ingredients to produce a feed additive composition for supplementing the foodstuff with the phytase;
mixing the feed additive composition with the foodstuff; and feeding the animal the foodstuff supplemented with the feed additive composition.

4. A method of improving the nutritional value of a foodstuff consumed by an avian species wherein the foodstuff increases the bone mass and bone mineral content of the avian species by increasing the bioavailability of phosphate from phytate wherein the foodstuff comprises myo-inositol hexakisphosphate, the method comprising the step of feeding to the avian species the foodstuff in combination with less than 1200 units of an E. coli 6-phytase expressed in yeast per kilogram of the foodstuff and an encapsulating agent, wherein the bioavailability of phosphate from phytate is increased by at least 1.5-fold compared to the bioavailability of phosphate from phytate obtained by feeding to a non-avian species the foodstuff in combination with the E. coli 6-phytase expressed in yeast.

5. A method of improving the nutritional value of a foodstuff consumed by an avian species wherein the foodstuff comprises myo-inositol hexakisphosphate, the method comprising the step of feeding to the avian species the foodstuff in combination with an E. coli 6-phytase expressed in yeast and an encapsulating agent wherein the bone mass and mineral content of the avian species is increased.

6. A method of improving the nutritional value of a foodstuff consumed by an avian species wherein the foodstuff increases the bone mass and bone mineral content of the animal, and wherein the foodstuff comprises myo-inositol hexakisphosphate, the method comprising the step of feeding to the avian species the foodstuff in combination with an E. coli 6-phytase expressed in yeast and an encapsulating agent wherein the number of eggs laid and the weight of the eggs laid by the avian species is increased.

7. The method of claim 1 or 2 wherein the animal is an avian species.

8. The method of claim 4 or 7 wherein the avian species is selected from the group consisting of a chicken, a turkey, a duck, and a pheasant.

9. The method of claim 1 or 2 wherein the animal is a marine or a fresh water aquatic species.

10. The method of claim 1 or 2 wherein the animal is a domestic animal.

11. The method of claim 10 wherein the domestic animal is a canine species.

12. The method of claim 10 wherein the domestic animal is a feline species.

13. The method of claim 1 or 2 wherein the animal is a human.

14. The method of claim 8 wherein the foodstuff is poultry feed.

15. The method of any one of claims 1, 2 and 4 wherein the yeast is selected from the group consisting of Saccharomyces species, Pichia species, Kluyveromyces species, Hansenula species, and Candida species.

16. The method of claim 1 or 2 wherein the yeast is Saccharomyces cerevisiae.

17. The method of claim 1 or 2 wherein the yeast is Pichia pastoris.

18. The method of any one of claims 1, 2 and 4 wherein the animal is fed the foodstuff in combination with from about 50 to about 1000 units of the phytase expressed in yeast per kilogram of the foodstuff.

19. The method of any one of claims 1, 2 and 4 wherein the animal is fed the foodstuff in combination with from about 50 to about 700 units of the phytase expressed in yeast per kilogram of the foodstuff.

20. The method of any one of claims 1, 2 and 4 wherein the animal is fed the foodstuff in combination with from about 50 to about 500 units of the phytase expressed in yeast per kilogram of the foodstuff.

21. The method of any one of claims 1, 2 and 4 wherein the animal is fed the foodstuff in combination with from about 50 to about 200 units of the phytase expressed in yeast per kilogram of the foodstuff.

22. The method of any one of claims 1, 2 and 4 wherein the phytase has an optimal activity at a pH of less than about 4.

23. The method of claim 2 wherein the animal is fed the foodstuff in combination with less than 1200 units of the phytase expressed in yeast per kilogram of the foodstuff.

24. The method of claim 2 wherein the phytase expressed in yeast has an amino acid sequence as specified in SEQ ID No.: 5.

25. The method of claim 24 wherein the phytase expressed in yeast differs from wild type AppA by at least one amino acid substitution which disrupts disulfide bond formation between cysteine residues at positions 100 and 210.

26. The method of claim 1 wherein the animal a porcine species.

27. The method of claim 2 wherein the animal is a pig.

28. The method of claim 26 or 27 wherein the foodstuff is pig feed.

29. The method of claim 1 or 4 wherein the phytase expressed in yeast is cleaved with a protease to enhance the capacity of the phytase to increase the bioavailability of phosphate from phytate compared to intact yeast-expressed phytase.

30. The method of claim 2 wherein the phytase expressed in yeast is cleaved with a protease to enhance the capacity of the phytase to increase the bone mass and mineral content of the animal compared to intact yeast-expressed phytase.

31. The method of claim 4 wherein the animal is fed the foodstuff in combination with 2000 units or less of the phytase expressed in yeast per kilogram of the foodstuff.

32. The method of claim 4 wherein the animal is fed the foodstuff in combination with 1500 units or less of the phytase expressed in yeast per kilogram of the foodstuff.

33. The method of claim 4 wherein the bioavailability of phosphate from phytate obtained by feeding the foodstuff in combination with the phytase expressed in yeast is increased by at least 2-fold compared to the bioavailability of phosphate from phytate obtained by feeding the foodstuff in combination with the same units of a phytase expressed in a non-yeast host cell.

34. The method of claim 5 wherein the bone mass and mineral content of the avian species obtained by feeding to the avian species the foodstuff in combination with the phytase expressed in yeast is increased by at least 1. 5-fold compared to the bone mass and mineral content obtained by feeding to a non-avian species the foodstuff in combination with the phytase expressed in yeast.

35. The method of claim 3 wherein the foodstuff is supplemented with less than 1200 units of the phytase per kilogram of the foodstuff and wherein the bioavailability to the animal of phosphate from phytate is increased by at least 2-fold compared to the bioavailability of phosphate from phytate obtained by feeding the foodstuff in combination with the same units of a phytate expressed in a non-yeast host cell.

36. Use of foodstuff comprising myo-inositol hexakisphosphate in combination with an E. coli 6-phytase expressed in yeast and an encapsulating agent, for the reduction of the feed to weight gain ratio of a monogastric animal wherein the foodstuff increases the bone mass and bone mineral content of the animal.

37. Use of a foodstuff comprising myo-inositol hexakisphosphate in combination with an E. coli 6-phytase expressed in yeast and an encapsulating agent, for the reduction of the feed to weight gain ratio of an avian species wherein the foodstuff increases the bone mass and bone mineral content of the animal.

38. The use of claim 36 wherein the animal is an avian species.

39. The use of claim 38 wherein the avian species is selected from the group consisting of a chicken, a turkey, a duck, and a pheasant.

40. The use of claim 36 wherein the animal is a marine or a fresh water aquatic species.

41. The use of claim 36 wherein the animal is a domestic animal.

42. The use of claim 41 wherein the domestic animal is a canine species.

43. The use of claim 41 wherein the domestic animal is a feline species.

44. The use of claim 36 wherein the animal is a human.

45. The use of claim 39 wherein the foodstuff is poultry feed.

46. The use of claim 36 wherein the yeast is selected from the group consisting of Saccharomyces species, Pichia species, Kluyveromyces species, Hansenula species, and Candida species.

47. The use of claim 36 wherein the yeast is Saccharomyces cerevisiae.

48. The use of claim 36 wherein the yeast is Pichia pastoris.

49. The use of claim 36 wherein the animal is fed the foodstuff in combination with from about 50 to about 1000 units of the phytase expressed in yeast per kilogram of the foodstuff.

50. The use of claim 36 wherein the animal is fed the foodstuff in combination with from about 50 to about 700 units of the phytase expressed in yeast per kilogram of the foodstuff.

51. The use of claim 36 wherein the animal is fed the foodstuff in combination with from about 50 to about 500 units of the phytase expressed in yeast per kilogram of the foodstuff.

52. The use of claim 36 wherein the animal is fed the foodstuff in combination with from about 50 to about 200 units of the phytase expressed in yeast per kilogram of the foodstuff.

53. The use of claim 36 wherein the phytase has an optimal activity at a pH of less than about 4.

54. The use of claim 36 wherein the animal is fed the foodstuff in combination with less than 1200 units of the phytase expressed in yeast per kilogram of the foodstuff.

55. The use of claim 36 wherein the phytase expressed in yeast has an amino acid sequence as specified in SEQ ID NO: 5.

56. The use of claim 55 wherein the phytase expressed in yeast differs from wild type AppA by at least one amino acid substitution which disrupts disulfide bond formation between cysteine residues at positions 100 and 210.

57. The use of claim 36 wherein the animal is a pig.

58. The use of claim 57 wherein the foodstuff is pig feed.

59. The use of claim 36 wherein the phytase expressed in yeast is cleaved with a protease to enhance the capacity of the phytase to reduce the feed to weight gain ratio of the animal compared to intact yeast-expressed phytase.

60. A feed additive composition for addition to an animal feed comprising a yeast-expressed E. coli 6-phytase, an encapsulating agent, and a carrier for the phytase, for use to increase the bone mass and bone mineral content of the animal, wherein the concentration of the phytase in the feed additive composition is greater than the concentration of the phytase in the final feed mixture.

61. The feed additive composition of claim 60 wherein the phytase is spray dried.

62. The feed additive composition of claim 60 wherein the phytase is selected from the group consisting of Escherichia coli-derived AppA2 and a site-directed mutant of Escherichia coli-derived AppA.

63. The feed additive composition of claim 60 wherein the carrier is selected from the group consisting of rice hulls, wheat middlings, vegetable fat, a hydrogenated lipid, a polysaccharide, a monosaccharide, mineral oil, calcium carbonate, gelatin, milk powder, phytate and other phytate-containing compounds, and a base mix.

64. The feed additive composition of claim 63 wherein the base mix comprises vitamins and minerals.

65. A foodstuff comprising the feed additive composition of claim 60 wherein the concentration of the phytase in the final feed mixture is less than 1200 units of the phytase per kilogram of the final feed mixture.

66. The foodstuff of claim 65 wherein the final feed mixture comprises from about 50 to about 1000 units of the phytase expressed in yeast per kilogram of the final feed mixture.

67. The foodstuff of claim 65 wherein the final feed mixture comprises from about 50 to about 700 units of the phytase expressed in yeast per kilogram of the final feed mixture.

68. The foodstuff of claim 65 wherein the final feed mixture comprises from about 50 to about 500 units of the phytase expressed in yeast per kilogram of the final feed mixture.

69. The foodstuff of claim 65 wherein the final feed mixture comprises from about 50 to about 200 units of the phytase expressed in yeast per kilogram of the final feed mixture.

70. The foodstuff of claim 65 wherein the final feed mixture further comprises 0.1 % exogenously added inorganic phosphate or less.

71. The foodstuff of claim 65 wherein the foodstuff exhibits increased nutritional value compared to the nutritional value of a foodstuff containing the same units of a phytase expressed in a non-yeast host cell.