Heat tolerant phytases

Details Heat tolerant phytases Heat tolerant phytases

2000-08-09

2004-03-02

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S196000, C435S254110, C435S254200, C435S320100, C435S018000, C435S069100, C435S255100, C435S325000, C536S023200

Reexamination Certificate

active

06699704

ABSTRACT:

BACKGROUND OF THE INVENTION
Phosphorus is an essential element for the growth of all organisms. In livestock production, feed must be supplemented with inorganic phosphorus in order to obtain a good growth is performance of monogastric animals (for example, pigs, poultry and fish).
In contrast, no inorganic phosphate needs to be added to the feedstuffs of ruminant animals. Microorganisms, present in the rumen, produce enzymes which analyze the conversion of phytate (myo-inositolhexakis-phosphate) to inositol and inorganic phosphate.
Phytate occurs as a storage phosphorus source in virtually all feed substances originating from plants. Phytate comprises 1-3% of all nuts, cereals, legumes, oil seeds, spores and pollen. Complex salts of phytic acid are termed phytin. Phytic acid is considered to be an anti-nutritional factor since it chelates minerals such as calcium, zinc, magnesium, iron and may also react with proteins, thereby decreasing the bioavailability of protein and nutritionally important minerals.
Phytate phosphorous passes through the gastro-intestinal tract of monogastric animals and is excreted in the manure. Though some hydrolysis of phytate does occur in the colon, the thus-released inorganic phosphorus has no nutritional value since inorganic phosphorus is absorbed only in the small intestine. As a consequence, a significant amount of the nutritionally important phosphorus is not used by monogastric animals, despite its presence in the feed.
The excretion of phytate phosphorus in manure has further consequences. Intensive livestock production has increased enormously during the past decades. Consequently, the amount of manure produced has increased correspondingly and has caused environmental problems in various parts of the world. This is due, in part, to the accumulation of phosphate from manure in surface waters which has caused eutrophication. For other background information, see European Patent Application Publication No. 420 358.
Phytases (myo-inositol hexakisphosphate phosphohydrolases; EC 3.1.3.8) are enzymes that hydrolyze phytate (myo-inositol hexakisphosphate) to myo-inositol and inorganic phosphate and are known to be valuable feed additives.
A phytase was first described in rice bran in 1907 [Suzuki et al., Bull. Coll. Agr. Tokyo Imp. Univ. 7, 495 (1907)] and phytases from Aspergillus species in 1911 [Dox and Golden, J. Biol. Chem. 10, 183-186 (1911)]. Phytases have also been found in wheat bran, plant seeds, animal intestines and in microorganisms [Howsen and Davis, Enzyme Microb. Technol. 5, 377-382 (1983), Lambrechts et al., Biotech. Lett. 14, 61-66 (1992), Shieh and Ware, Appl. Microbiol. 16, 1348-1351 (1968)].
The cloning and expression of the phytase from
Aspergillus niger
(ficuum) has been described by Van Hartingsveldt et al., in Gene, 127, 87-94 (1993) and in European Patent Application, Publication No. 420 358 and from
Aspergillus niger
var awamori by Piddington et al. in Gene 133, 55-62 (1993).
Since phytases used so far in agriculture have certain disadvantages, it is an object of the present invention to provide new phytases or polypeptides having phytase activity with improved properties. Since it is known that phytases used so far lose activity during feed pelleting process due to heat treatment, improved heat tolerance would be such an improved property.
So far, phytases have not been reported in thermotolerant fungus with the exception of
Aspergillus fumigatus
[Dox and Golden et al., J. Biol. Chem. 10, 183-186 (1911)] and
Rhizopus oryzae
[Howson and Davies, Enzyme Microb. Technol. 5, 377-382 (1993)]. Thermotolerant phytases have been described originating from
Aspergillus terreus
Strain 9A-1 [Temperature optimum 70° C.; Yamada et al., Agr. Biol. Chem. 32, 1275-1282 (1968)] and
Schwanniomyces castellii
[Temperature optimum 77° C.; Segueilha et al., Bioeng. 74, 7-11 (1992)]. However for commercial use in agriculture such phytases must be available in large quantities. Accordingly it is an object of the present invention to provide DNA sequences coding for heat tolerant phytases. Improved heat tolerance of phytases encoded by such DNA sequences can be determined by assays known in the art, for example, by the processes used for feed pelleting or assays determining the heat dependence of the enzymatic activity itself as described, for example, by Yamada et al. (s.a.).
It is furthermore an object of the present invention to screen fungi which show a certain degree of thermotolerance for phytase production. Such screening can be made as described, for example, in Example 1. In this way heat tolerant fungal strains, listed in Example 1, have been identified for the first time to produce a phytase.
Heat tolerant fungal strains, see for example, those listed in Example 1, can then be grown as known in the art, for example, as indicated by their supplier, for example, the American Tissue Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Agricultural Research Service Culture Collection (NRRL) and the Centralbureau voor Schimmelcultures (CBS) from which such strains are available or as indicated, for example, in Example 2.
Further improved properties are, for example, an improved substrate specificity regarding phytic acid [myo-inositol(1,2,3,4,5,6)hexakisphosphate] which is a major storage form of phosphorous in plants and seeds. Since for the complete release of the six phosphate groups from phytic acid, a phytase and a pH 2.5. acid phosphatase activity are required, a polypeptide having phytase and pH 2.5 acid phosphatase activity would be highly desirable. For example, International Patent Application Publication No. 94/03072 discloses an expression system which allows the expression of a mixture of phytate degrading enzymes in desired ratios. However, it would be even more desirable to have both such activities in a single polypeptide. Therefore it is also an object of the present invention to provide DNA sequences coding for such polypeptides. Phytase and phosphatase activities can be determined by assays known in the state of the art or described, for example, in Example 9.
Another improved property is, for example, a so called improved pH-profile. This means, for example, two phytin degrading activity maxima, for example, one at around pH 2.5 which could be the pH in the stomach of certain animals and another at around pH 5.5 which could be the pH after the stomach in certain animals. Such pH profile can be determined by assays known in the state of the art or described, for example, in Example 9. Accordingly it is also an object of the present invention to provide DNA sequences coding for such improved polypeptides.
It is yet another object of the present invention to provide a DNA sequence coding for a polypeptide having phytase activity and which DNA sequence is derived from a fungus selected from the group consisting of
Acrophialophora levis, Aspergillus terreus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus sojae, Calcarisporiella thermophila, Chaetomium rectopilium, Corynascus thermophilus
, Humicola sp.,
Mycelia sterilia, Myrococcum thermophilum, Myceliophthora thermophila, Rhizomucor miehei, Sporotrichum cellulophilum, Sporotrichum thermophile, Scytalidium indonesicum
and
Talaromyces thermophilus
or a DNA sequence coding for a fragment of such a polypeptide which fragment still has phytase activity, or more specifically such a DNA sequence wherein the fungus is selected from the group consisting of
Acrophialophora levis, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus, Calcarisporiella thermophila, Chaetomium rectopilium, Corynascus thermophilus, Sporotrichum cellulophilum, Sporotrichum thermophile, Mycelia sterilia, Myceliophthora thermophila
and
Talaromyces thermophilus
, or more specifically such a DNA sequence wherein the fungus is selected from the group consisting of
Aspergillus terreus, Myceliophthora thermophila, Aspergillus fumigatus, Aspergil

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