Phytase polypeptides

Details Phytase polypeptides Phytase polypeptides

2000-01-13

2003-05-27

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S069100, C435S183000, C435S252300, C435S320100, C536S023100, C536S023200, C536S023600, C536S024330

Reexamination Certificate

active

06569659

ABSTRACT:

FIELD OF INVENTION
The present invention relates to isolated polypeptides having phytase activity, the corresponding cloned DNA sequences, a method of producing such polypeptides, and the use thereof for a number of industrial applications. In particular, the invention relates to phytases derived from the phyllum Basidiomycota, phytases of certain consensus sequences and fungal 6-phytases.
BACKGROUND OF THE INVENTION
Phytic acid or myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate (or for short myo-inositol hexakisphosphate) is the primary source of inositol and the primary storage form of phosphate in plant seeds. In fact, it is naturally formed during the maturation of seeds and cereal grains. In the seeds of legumes it accounts for about 70% of the phosphate content and is structurally integrated with the protein bodies as phytin, a mixed potassium, magnesium and calcium salt of inositol. Seeds, cereal grains and legumes are important components of food and feed preparations, in particular of animal feed preparations. But also in human food cereals and legumes are becoming increasingly important.
The phosphate moieties of phytic acid chelates divalent and trivalent cations such as metal ions, i.a. the nutritionally essential ions of calcium, iron, zinc and magnesium as well as the trace minerals mangane, copper and molybdenum.
Besides, the phytic acid also to a certain extent binds proteins by electrostatic interaction. At a pH below the isoelectric point, pI, of the protein, the positively charged protein binds directly with phytate. At a pH above pI, the negatively charged protein binds via metal ions to phytate.
Phytic acid and its salts, phytates, are often not metabolized, since they are not absorbable from the gastro intestinal system, i.e. neither the phosphorous thereof, nor the chelated metal ions, nor the bound proteins are nutritionally available.
Accordingly, since phosphorus is an essential element for the growth of all organisms, food and feed preparations need to be supplemented with inorganic phosphate. Quite often also the nutritionally essential ions such as iron and calcium, must be supplemented. And, besides, the nutritional value of a given diet decreases, because of the binding of proteins by phytic acid. Accordingly, phytic acid is often termed an anti-nutritional factor.
Still further, since phytic acid is not metabolized, the phytate phosphorus passes through the gastrointestinal tract of such animals and is excreted with the manure, resulting in an undesirable phosphate pollution of the environment resulting e.g. in eutrophication of the water environment and extensive growth of algae.
Phytic acid or phytates, said terms being, unless otherwise indicated, in the present context used synonymously or at random, are degradable by phytases.
In most of those plant seeds which contain phytic acid, endogenous phytase enzymes are also found. These enzymes are formed during the germination of the seed and serve the purpose of liberating phosphate and, as the final product, free myo-inositol for use during the plant growth.
When ingested, the food or feed component phytates are in theory hydrolyzable by the endogenous plant phytases of the seed in question, by phytases stemming from the microbial flora in the gut and by intestinal mucosal phytases. In practice, however the hydrolyzing capability of the endogenous plant phytases and the intestinal mucosal phytases, if existing, is far from sufficient for increasing significantly the bioavailibility of the bound or constituent components of phytates. However, when the process of preparing the food or feed involve germination, fermentation or soaking, the endogenous phytase might contribute to a greater extent to the degradation of phytate.
In ruminant or polygastric animals such as horses and cows the gastro intestinal system hosts microorganisms capable of degrading phytic acid. However, this is not so in monogastric animals such as human beings, poultry and swine. Therefore, the problems indicated above are primarily of importance as regards such monogastric animals.
The production of phytases by plants as well as by microorganisms has been reported. Amongst the microorganisms, phytase producing bacteria as well as phytase producing fungi are known.
From the plant kingdom, e.g. a wheat-bran phytase is known (Thomlinson et al, Biochemistry, 1 (1962), 166-171). An alkaline phytase from lilly pollen has been described by Barrientos et al, Plant. Physiol., 106 (1994), 1489-1495.
Amongst the bacteria, phytases have been described which are derived from
Bacillus subtilis
(Paver and Jagannathan, 1982
, Journal of Bacteriology
151:1102-1108) and
Pseudomonas
(Cosgrove, 1970
, Australian Journal of Biological Sciences
23:1207-1220). Still further, a phytase from
E. coli
has been purified and characterized by Greiner et al,
Arch. Biochem. Biophys
., 303, 107-113, 1993). However, this enzyme is probably an acid phosphatase.
Phytase producing yeasts are also described, such as
Saccharomyces cerevisiae
(Nayini et al, 1984
, Lebensmittel Wissenschaft und Technologie
17:24-26. However, this enzyme is probably a myo-inositol monophosphatase (Wodzinski et al,
Adv. Appl. Microbiol
., 42, 263-303). AU-A-24840/95 describes the cloning and expression of a phytase of the yeast
Schwanniomyces occidentalis.
There are several descriptions of phytase producing filamentous fungi, however only belonging to the fungal phyllum of Ascomycota (ascomycetes). In particular, there are several references to phytase producing ascomycetes of the Aspergillus genus such as
Aspergillus terreus
(Yamada et al., 1986
, Agric. Biol. Chem
. 322:1275-1282). Also, the cloning and expression of the phytase gene from
Aspergillus niger
var.
awamori
has been described (Piddington et al., 1993
, Gene
133:55-62). EP 0 420 358 describes the cloning and expression of a phytase of
Aspergillus ficuum
(
niger
). EP 0 684 313 describes the cloning and expression of phytases of the ascomycetes
Myceliophthora thermophila
and
Aspergillus terreus.
NOMENCLATURE AND POSITION SPECIFICITY OF PHYTASES
In the present context a phytase is an enzyme which catalyzes the hydrolysis of phytate (myo-inositol hexakisphosphate) to (1) myo-inositol and/or (2) mono-, di-, tri-, tetra- and/or 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 P+one or more of the components IP5, IP4, IP3, IP2, IP1 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
. For convenience Ins(1,2,3,4,5,6)P
6
(phytic acid) is abbreviated PA.
According to the Enzyme nomenclature database ExPASy (a repository of information relative to the nomenclature of enzymes primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) describing each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided), two different types of phytases are known: A so-called 3-phytase (miyo-inositol hexaphosphate 3-phosphohydrolase, EC 3.1.3.8) and a so-called 6-phytase (myo-inositol hexaphosphate 6-phosphohydrolase, EC 3.1.3.26). The 3-phytase hydrolyses first the ester bond at the 3-position, whereas the 6-phytase hydrolyzes first the ester bond at the 6-position.
Inositolphosphate Nomenclature
Considering the primary hydrolysis products of a phytase acting on phytic acid, some of the resulting esters are diastereomers and some are enantiomers. Generally, it is easier to discriminate between diastereomers, since they have different physical properties, whereas it is much more difficult to discriminate between enantiomers which are mirror images of each other.
Thus, Ins(1,2,4,5,6)P
5
(3-phosphate removed) and Ins(1,2,3,4,5)P
5
(6-phosphate removed) are diastereomers and easy to discriminate, whereas Ins(1,

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