Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-09-30
2001-04-24
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C436S006000, C424S094600
Reexamination Certificate
active
06221644
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to isolated polypeptides having phytase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the polypeptides. The invention further relates to compositions comprising the polypeptides and methods of use thereof.
2. Description of the Related Art
Phytases (myo-inositol hexakisphosphate phosphohydrolases, EC 3.1.3.8) catalyze the hydrolysis of phytate (myo-inositol hexakisphosphate) to (1) myo-inositol, (2) mono-, di-, tri-, tetra- and penta-phosphates thereof and (3) inorganic phosphate. In the following, for short, the above compounds are sometimes referred to as IP6, I, IP1, IP2, IP3, IP4, IP5 and P, respectively. This means that by action of a phytase, IP6 is degraded into inorganic phosphate and one or more of the components IP5, IP4, IP3, IP2, IP11 and I. Alternatively, myo-inositol carrying in total n phosphate groups attached to positions p, q, r, . . . is denoted (Ins(p,q,r,. . .)P
n
).
Two different types of phytases are known: A so-called 3-phytase (myo-inositol hexakisphosphate 3-phosphohydrolase, EC 3.1.3.8) and a so-called 6-phytase (myo-inositol hexakisphosphate 6-phosphohydrolase, EC 3.1.3.26). The 3-phytase hydrolyzes first the ester bond at the 3-position, whereas the 6-phytase hydrolyzes first the ester bond at the 6-position. The remaining ester bonds of the resulting IP5 substrate (whether the 1,2,4,5,6-IP5 or the 1,2,3,4,5-IP5) are subsequently hydrolyzed at different rates. Also the rate of hydrolysis of the components IP4, IP3, IP2 and IP1 seems to be variable, if hydrolyzed at all.
Phytase-producing microorganisms include bacteria such as
Bacillus subtilis
(Paver and Jagannathan, 1982,
Journal of Bacteriology
151: 1102-1108) and Pseudomonas (Cosgrove, 1970, Australian Journal of Biological Sciences 23: 1207-1220); yeast such as
Saccharomyces cerevisiae
(Navini and Marcakis, 1984,
Lebensmittei Wissenschaft und Technologie
17: 24-26; and fungi of the Aspergillus genus such as
Aspergillus terreus
(Yamada et al., 1986,
Agricultural Biological Chemistry
322: 1275-1282).
The cloning and expression of the phytase genes from
Aspergillus niger
var.
awamori
by Piddington et al. (1993,
Gene
133: 55-62) and
Aspergillus niger
(
ficuum
) by van Hartingsveldt et al. (1993,
Gene
127: 87-94; EP 420 358) have been disclosed.
Phytic acid is the primary storage form of phosphate in cereal grains, legumes, and oilseeds, such as soy, which are the principal components of animal feeds. However, the presence of phytic acid in animal feeds for monogastric animals is undesirable because the phosphate moieties of phytic acid chelate essential minerals and possibly proteins making them nutritionally unavailable. Furthermore, phytate phosphorus passes through the gastrointestinal tract of monogastric animals and is not metabolized. Since phosphorus is an essential element for the growth of all organisms, livestock feed must be supplemented with inorganic phosphate. Thus, the art has described the use of phytases in feeds of monogastric animals.
Furthermore, since phytic acid is not metabolized by monogastric animals, it is excreted in manure. The amount of manure produced worldwide has increased significantly resulting from increased livestock production. The disposal of manure has caused an environmental problem in various locations around the world due to the accumulation of phosphate particularly in water. Thus, the art has also described the use of phytases for reducing the amount of phytate in manure.
There is a need in the art for new phytases with improved properties which can be produced in commercially significant quantities.
It is an object of the present invention to provide a new class of phytases, i.e., 3,6-phytases, i.e., phytases which attack both bonds of a phosphoester.
SUMMARY OF THE INVENTION
The present invention relates to isolated polypeptides having phytase activity selected from the group consisting of:
(a) a polypeptide having 3,6-phytase activity;
(b) a polypeptide with an amino acid sequence which has at least 60% identity with the amino acid sequence set forth in SEQ ID NO:2;
(c) a polypeptide which is encoded by a nucleic acid sequence which is capable of hybridizing under medium stringency conditions with (i) the nucleic acid sequence set forth in SEQ ID NO: 1 or (ii) its complementary strand;
(d) an allelic form of (b) or (c); and
(e) a fragment of (b), (c), or (d).
The present invention also relates to isolated nucleic acid sequences encoding the polypeptides and to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the polypeptides. The present invention further relates to composite feeds and methods of reducing phytate levels.
REFERENCES:
patent: 5866118 (1999-02-01), Berka et al.
patent: 0 420 358 A1 (1991-04-01), None
patent: 0 684 313 A2 (1995-11-01), None
patent: WO 96/28567 (1996-09-01), None
Knuckles et al. (1987) Journal of Food Science 52(3) :719-721.
Berka Randy M.
Klotz Alan V.
Rey Michael W.
Achutamurthy Ponnathapu
Lambiris Esq. Elias
Novo Nordisk Biotech Inc.
Starnes Robert L.
Tung Peter P.
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