Phytase

Details Phytase Phytase

1999-04-13

2001-02-20

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S320100, C435S325000, C435S252100, C536S023100, C536S023200

Reexamination Certificate

active

06190897

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production and isolation of such polynucleotides and polypeptides. More particularly, the polypeptides of the present invention have been identified as phytases and in particular, enzymes having phytase activity.
BACKGROUND
Minerals are essential elements for the growth of all organisms. For livestock production of monogastric animals (e.g., pigs, poultry) and fish, feed is commonly supplemented with minerals. Plant seeds are a rich source of minerals since they contain ions that are complexed with the phosphate groups of phytic acid. Ruminants do not require inorganic phosphate and minerals because microorganisms in the rumen produce enzymes that catalyze conversion of phytate (myo-inositol-hexaphosphate) to inositol and inorganic phosphate. In the process, minerals that have been complexed with phytate are released.
Phytate occurs as a source of stored phosphorous in virtually all plant feeds (Phytic Acid, Chemistry and Applications, E. Graf (Ed.), Pilatus Press: Minneapolis, Minn. U.S.A., 1986). Phytic acid forms a normal part of the seed in cereals and legumes. It functions to bind dietary minerals that are essential to the new plant as it emerges from the seed. When the phosphate groups of phytic acid are removed by the seed enzyme phytase, the ability to bind metal ions is lost and the minerals become available to the plant. In livestock feed grains, the trace minerals bound by phytic acid are only partially available for absorption by monogastric animals, which lack phytase activity. Although some hydrolysis of phytate occurs in the colon, most phytate passes through the gastrointestinal tract of monogastric animals and is excreted in the manure contributing to fecal phosphate pollution problems in areas of intense livestock production. Inorganic phosphorous released in the colon has no nutritional value to livestock because inorganic phosphorous is absorbed only in the small intestine. Thus, a significant amount of the nutritionally important dietary minerals are potentially not available to monogastric animals.
Conversion of phytate to inositol and inorganic phosphorous can be catalyzed by microbial enzymes referred to broadly as phytases. Phytases such as phytase #EC 3.1.3.8 are capable of catalyzing hydrolysis of myo-inositol hexaphosphate to D-myo-inositol 1,2,4,5,6-pentaphosphate and orthophosphate. Certain fungal phytases reportedly hydrolyze inositol pentaphosphate to tetra-, tri-, and lower phosphates; e.g.,
A. ficuum
phytases reportedly produce mixtures of myoinositol di- and mono-phosphate (Ullah, 1988). Phytase producing microorganisms comprise bacteria such as
Bacillus subtilis
(V. K. Powar and V. J. Jagannathan, J. Bacteriol. 151:1102-1108, 1982) and Pseudomonas (D. J. Cosgrove, Austral. J. Biol. Sci. 2:1207-1220, 1970); yeasts such as
Sacchoromyces cerevisiae
(N. R. Nayini and P. Markakis, Lebensmittel Wissenschaft und Technologie 17:24-26, 1984); and fungi such as
Aspergillus terreus
(K. Yamada, et al., Agric. Biol Chem. 32:1275-1282, 1968). The possible use of microbes capable of producing phytase as a feed additive for monogastric animals has been reported previously (Shieh and Ware, U.S. Pat. No.3,297,548; Nelson, T. S. et al., J. Nutrition 101:1289-1294, 1971).
Microbial phytases may also reportedly be useful for producing animal feed from certain industrial processes, e.g., wheat and corn waste products. The wet milling process of corn produces glutens sold as animal feeds. Addition of phytase may reportedly improve the nutritional value of the feed product. Fungal phytase enzymes and process conditions (t~50° C. and pH ~5.5) have been reported previously in European Patent Application 0 321 004. In processing soybean meal the presence of phytate reportedly renders the meal and wastes unsuitable for feeds used in rearing fish, poultry and other non-ruminants as well as calves fed on milk. Phytase is reportedly useful for improving the nutrient and commercial value of this high protein soy material (see Finase Enzymes by Alko, Rajamaki, Finland). A combination of phytase and a pH 2.5 optimum acid phosphatase form
A. niger
has been used by Alko, Ltd as an animal feed supplement in their phytic acid degradative product Finas F and Finase S. A cost-effective source of phytase would greatly enhance the value of soybean meals as an animal feed (Shieh et al., 1969).
Phytase and less specific acid phosphatases are produced by the fungus
Aspergillus ficuum
as extracellular enzymes (Shieh et al., 1969). Ullah reportedly purified a phytase from wild-type
A. ficuum
that had an apparent molecular weight of 61.7 kDA (on SDS-PAGE; as corrected for glycosylation); pH optima at pH 2.5 and pH 5.5; a Km of about 40 &mgr;m; and, a specific activity of about 50 U/mg (Ullah, A., Preparative Biochem 18:443-458, 1988); PCT patent application WO 91/05053 also reportedly discloses isolation and molecular cloning of a phytase from
Aspergillus ficuum
with pH optima at pH 2.5 and pH 5.5, a Km of about 250 &mgr;m, and specific activity of about 100 U/mg protein.
Acid phosphatases are enzymes that catalytically hydrolyze a wide variety of phosphate esters and usually exhibit pH optima below 6.0 (Hollander, 1971); e.g., #EC 3.1.3.2 catalyzes hydrolysis of orthophosphoric monoesters to orthophosphate products. An acid phosphatase has reportedly been purified from
A. ficuum
. The deglycosylated form of the acid phosphatase has an apparent molecular weight of 32.6 kDa (Ullah et al., 1987).
The object of the present invention provides a recombinant phytase isolated from
Escherichia coli
B that improves the efficiency of release of phosphorous from phytate and the salts of phytic acid. Another object of the present invention provides a source of a recombinant enzyme that is suitable for commercial use in feeds and industrial processes with minimal processing.
SUMMARY OF THE INVENTION
The present invention provides a polynucleotide and a polypeptide encoded thereby which has been identified as a phytase enzyme having phytase activity. In accordance with one aspect of the present invention, there is provided a novel recombinant enzyme, as well as active fragments, analogs and derivatives thereof.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the enzyme of the present invention including mRNA, DNA, cDNA, genomic DNA as well as active analogs and fragments of such enzyme.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptide by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding an enzyme of the present invention, under conditions promoting expression of said enzyme and subsequent recovery of said enzyme.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such enzyme, or polynucleotide encoding such enzymes for use in commercial processes, such as, for example, processes that liberate minerals from phytates in plant materials either in vitro, i.e., in feed treatment processes, or in vivo, i.e., by administering the enzyme to animals.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such enzymes, or polynucleotides encoding such enzymes, for in vitro purposes related to scientific research, for example, to generate probes for identifying similar sequences which might encode similar enzymes from other organisms.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.


REFERENCES:
patent: 5366736 (1994-11-01), Edwards, Jr.
patent: 5436156 (1995-07-01), Van Gorcom et al.
patent: 5939303 (1999-08-01), Cheng et al.
Greiner, R. et al., Arch. Biochem. Biophys. vol. 303

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