providing a foodstuff comprising myo-inositol hexakisphosphate;providing a mutant phytase polypeptide, feeding to the animal the foodstuff in combination with the mutant phytase under conditions effective to increase the bioavailability of phosphate from phytate
Inventors
Lei, Xingen • Mullaney, Edward J. • Ullah, Abul
Assignees
Cornell Research Foundation Inc • US Department of Agriculture USDA
Publication Number
US-7736680-B2
Publication Date
2010-06-15
Expiration Date
2027-12-07
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Abstract
The present invention relates to an isolated nucleic acid molecule encoding a mutant phytase and the isolated mutant phytase itself. The present invention further relates to methods of using the isolated nucleic acid molecule and the isolated mutant phytase of the present invention.
Core Innovation
The invention relates to isolated nucleic acid molecules encoding mutant phytases from Aspergillus species, including Aspergillus niger and Aspergillus fumigatus, and isolated mutant phytase polypeptides. These mutant phytases contain amino acid substitutions at specific residues corresponding to those of wild-type phytases (e.g., residues 50, 91, 94, 228, 262, 300, and 301 of SEQ ID NO:2 for A. niger, and residue 363 of SEQ ID NO:4 for A. fumigatus). The invention also includes recombinant DNA expression systems, host cells, and methods for recombinantly producing and isolating these mutant phytases.
The mutant phytases exhibit altered and improved catalytic efficiency, modified substrate binding regions, and altered pH profiles and optima favoring performance in acidic environments such as the gastrointestinal tracts of animals. These improvements are achieved without sacrificing thermostability. The invention further relates to methods of feeding monogastric animals with foodstuffs combined with the mutant phytases to increase the bioavailability of phosphate from phytate and improve nutritional value, including in human foodstuffs.
The problem being solved is the inefficiency of native phytases, such as Aspergillus niger NRRL 3135 phyA, to effectively hydrolyze phytate phosphorus at the physiologically relevant acidic pH range found in animal stomachs (pH ~2.5 to 3.5). Native enzymes have pH activity dips and unique substrate specificity limitations that reduce phosphate bioavailability from plant-origin feed, particularly affecting monogastric animals that cannot utilize phytate phosphorus. There is a need for mutant phytases with enhanced catalytic activity, altered pH profiles to perform better under acidic conditions, and improved thermostability for use as animal feed additives and for improving mineral bioavailability in human nutrition.
Claims Coverage
The patent includes one independent claim defining isolated mutant phytases and several dependent claims detailing specific mutations and compositions.
Isolated mutant phytase with specific amino acid substitutions
An isolated mutant phytase comprising an amino acid sequence with at least 96 percent identity to SEQ ID NO:2 containing at least one amino acid residue substitution at residues 50, 91, 94, 262, 300, and/or 301, or having at least 96 percent identity to SEQ ID NO:4 with substitution at residue 363, exhibiting phytase activity.
Mutant phytase in pure, non-pure, or recombinant form
The isolated mutant phytase may be in pure, non-pure, or recombinant form.
Specific amino acid substitutions at defined residues
Substitutions include Q50L or Q50P at residue 50; K91A or K91E at 91; K94E at 94; D262H at 262; K300R, K300T, K300D, or K300E at 300; K301E at 301; and M363L at residue 363 of SEQ ID NO:4.
Composite substitutions including double, triple, and quadruple mutants
Claims include mutant phytases with multiple substitutions such as double substitutions K300E/K301E, triple substitutions K300E/K301E/K94E, and quadruple substitutions including K300D/K94A/E228A/D262A.
Animal feed compositions and foodstuffs containing mutant phytase
Compositions comprising the isolated mutant phytase for use in animal feed, foodstuffs optionally combined with vitamin and mineral mixes, soybean meal, and antibiotics.
Methods of feeding monogastric animals with foodstuff combined with mutant phytase
Feeding methods combining foodstuff and the isolated mutant phytase to animals including fowl, porcine, aquatic, domestic (canine, feline), and various mammals at specified phytase unit amounts per kilogram of foodstuff.
Methods of improving nutritional value by increasing phosphate bioavailability
Providing a foodstuff containing myo-inositol hexakisphosphate and feeding it with the mutant phytase to animals or humans under conditions effective to increase bioavailability of phosphate or minerals such as iron, zinc, phosphorus, and calcium.
The claims cover isolated mutant phytases with defined amino acid substitutions enhancing enzymatic properties, related compositions for animal feed and foodstuffs, and methods of use in feeding animals and improving mineral bioavailability.
Stated Advantages
Mutant phytases exhibit improved phytase activity over non-mutant phytases.
Mutant phytases have altered pH profiles and optima favoring use in acidic environments such as animal gastrointestinal tracts.
Improvements are achieved without sacrificing thermostability; mutant phytases have equal or better thermostability compared to non-mutant counterparts.
Mutant phytases may be useful for producing specific inositol phosphate metabolites for nutritional and biomedical applications.
Documented Applications
Use of mutant phytases as feed additives in animal feed compositions to improve phosphate digestion.
Feeding monogastric animals such as poultry, swine, and aquatic species with foodstuffs combined with mutant phytase to increase bioavailability of phosphate and minerals.
Incorporation of mutant phytases in human foodstuffs to enhance mineral (e.g., iron, zinc, phosphorus, calcium) bioavailability and improve nutrition.
Transgenic expression of mutant phytases in plants edible for animals to improve mineral nutritional value.
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