Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Food or edible as carrier for pharmaceutical
Reexamination Certificate
2001-06-22
2002-04-16
Naff, David M. (Department: 1651)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Food or edible as carrier for pharmaceutical
C424S094600, C426S807000, C435S195000, C435S196000
Reexamination Certificate
active
06372247
ABSTRACT:
TECHNICAL FIELD
The present invention relates to inexpensive phytases with low Michaelis constants (abbreviated hereinafter to Km) for phytic acid, which decompose phytic acid as anti-trophic factor contained in feed thereby improving the nutritive values of feed and simultaneously enabling efficient utilization of phosphate released by said decomposition.
BACKGROUND ART
Phosphorus is an element essential for every organism. Phosphorus is included in plant-derived feed used in breeding of domestic animals, and 50 to 70% of the phosphorus is present as phytic acid. Phytic acid occurring in a large amount in plant seeds is a major storage substance of phosphate. However, phytic acid is excreted without digestion and absorption in digestive organs in single-stomach animals such as pigs, chickens etc., so its phosphorus is not utilized at all although it is a major storage substance of phosphate. Accordingly, inorganic phosphate is added to feed for single-stomach animals for the purpose of growth promotion. However, addition of phosphate to feed results in an increase in the amount of phosphorus in feces. In recent years, as production of domestic animals is increased, feces from domestic animals are increased to cause an environmental problem in all over the world. In particular, phosphorus contained in feces is mentioned as a cause for the phenomenon of eutrophication in lakes and marshes, and the amount of excreted phosphorus comes to be regulated and there arises the necessity for dealing with it.
Further phytic acid chelates with divalent metals important as nutritious sources, such as magnesium, calcium, zinc, iron etc. to make them hardly adsorbed into animals, resulting in reduction of the nutritive values of feed. Accordingly, phytic acid is considered as an anti-trophic factor.
From the foregoing, improvements in the nutritive values of feed are attempted by treating feed with an enzyme for hydrolyzing phytic acid into inositol and inorganic phosphate thereby permitting the phytic acid to release the phosphate to substitute it for conventionally added phosphate whereby the amount of phosphorus in feces is decreased, and phytic acid as an anti-trophic factor is decomposed [U.S. Pat. No. 3,297,548 (1967), J. Nutrition 101, 1289-1294 (1971)]. Microorganisms known to produce phytase (enzyme decomposing phytic acid) include bacteria such as
Bacillus subtilis
and
Pseudomonas
, yeasts such as
Saccharomyces cerevisiae
, and filamentous fungi such as
Aspergillus terreus, Aspergillus ficuum
and
Aspergillus awamori
. With respect to the phytase derived from
Aspergillus ficuum
, its purification and biochemical properties are described in Preparative Biochem., 18, 443-458 (1988) and its gene and amino acid sequence are described in Gene, 127, 87-94 (1993). With respect to the phytase derived from
Aspergillus awamori
, its nucleotide sequence and amino acid sequence are described in Gene, 133, 55-62 (
1993).
In order to demonstrate the ability possessed by an enzyme, it is necessary for the concentration of its substrate to be higher than the Michaelis constant (Km), and in the case of enzymes having the same maximum reaction rate (Vmax), an enzyme having a lower Km value does not reduce the reaction rate even at lower substrate concentration as compared with an enzyme having a higher Km value. That is, an enzyme having a lower Km value can maintain the sufficient decomposition rate even at lower substrate concentration, and the amount of the substrate not decomposed can be minimized as compared with an enzyme having a higher Km value.
The Michaelis constants (Km) of known phytases derived from filamentous fungi are 250 &mgr;M for
Aspergillus ficuum
(WO 91/05053) and 330 &mgr;M
Aspergillus oryzae
(Biosci. Biotech. Biochem., 57, 1364-1365 (
1993)).
On one hand, acidic phosphatases are purified from various microorganisms and their properties are reported, and for example, 2 acidic phosphatases derived from
Aspergillus ficuum
are purified and their properties are examined [Prep. Biochem., 18, 37-65 (1988)]. However, said acidic phosphatases cannot use phytic acid as a substrate, so their utilization for the purpose of improving the nutritive values of feed as described above is not feasible.
Under the circumstances described above, there is a need for phytase which decomposes phytic acid as an anti-trophic factor contained in feed thereby improving the nutritive values of feed and simultaneously enabling efficient utilization of phosphate released by said decomposition.
DISCLOSURE OF THE INVENTION
Accordingly, the object of the present invention is to provide phytases having low Km values for phytic acid and a process for producing said phytases.
As a result of their extensive study for solving the problems described above, from microorganisms belonging to the genus Monascus, the present inventors found novel phytases having Km values of 10 to 110 &mgr;M when phytic acid was used as a substrate, and they revealed the properties thereof and stablished a process for producing said phytases to complete the present invention.
That is, the present invention relates to novel phytases having Km values of 10 to 110 &mgr;M and a process for producing said phytases.
Specific examples of the novel phytases of the invention include 3 phytases having the following physicochemical properties:
1. Phytase I
1) Km: 27 &mgr;M when phytic acid is used as a substrate;
2) optimum pH: pH 5.5;
3) pH stability: stable in the range of pH 5.5 to 6.5;
4) optimum temperature: 50° C.;
5) temperature stability: stable up to 35° C.;
6) substrate specificity: acting on phytic acid, p-nitrophenylphosphate, D-glucose-6-phosphate, fructose-6-phosphate, D-myo-inositol-2-phosphate, D-myo-inositol-1-phosphate, D-myo-inositol-1,4-diphosphate, and adenosine triphosphate as the substrate;
7) molecular weight: about 80 to 100 kDa (gel filtration method); and
8) isoelectric point: pI 5.7 (chromatofocusing method).
2. Phytase II
1) Km: 20 &mgr;M when phytic acid is used as a substrate;
2) optimum pH: pH 6.0;
3) pH stability: stable in the range of pH 6.0 to 7.0;
4) optimum temperature: 50° C.;
5) temperature stability: stable up to 50° C.;
6) substrate specificity: acting on phytic acid, p-nitrophenylphosphate, D-glucose-6-phosphate, fructose-6-phosphate, D-myo-inositol-2-phosphate, D-myo-inositol-1-phosphate, D-myo-inositol-1,4-diphosphate, and adenosine triphosphate as the substrate;
7) molecular weight: about 120 kDa (gel filtration method); and
8) isoelectric point: pI 4.8 (chromatofocusing method).
3. Phytase III
1) Km: 107 &mgr;M when phytic acid is used as a substrate;
2) optimum pH: pH 2.5;
3) pH stability: stable in the range of pH 2.0 to 8.0;
4) optimum temperature: 45° C.
5) temperature stability: stable up to 60 ° C.;
6) substrate specificity: acting on p-nitrophenylphosphate, phytic acid, D-glucose-6-phosphate, fructose-6-phosphate, D-myo-inositol-2-phosphate, D-myo-inositol-1-phosphate, D-myo-inositol-1,4-diphosphate, and adenosine triphosphate as the substrate;
7) molecular weight: about 140 kDa (gel filtration method); and
8) isoelectric point: pI 5.2 (chromatofocusing method); and
9) N-terminal amino acid sequence: shown in SEQ ID NO:1.
The microorganisms used in the present invention may be any microorganisms producing the novel phytases having Km values of 10 to 110 &mgr;M when phytic acid is used as a substrate, and examples are microorganisms belonging to the genus Monascus. Specifically,
Monascus anka
IFO 30873 can be mentioned. Further, animal cells having the ability to produce the novel phytases, which have Km values of 10 to 110 110 &mgr;M when phytic acid is used as a substrate, can also be used in the present invention.
The microorganism having the ablity to produce the novel phytase is culture in a conventional culture method until the novel phytase is formed and accumulated, and the novel phytase is recovered from the culture whereby the novel phytase can be produced. Hereinafter, the microorganism or mutant used for producing the novel phytase is called the
Anazawa Hideharu
Kato Yoko
Kuroyanagi Satoshi
Nagashima Tadashi
Sugimoto Seiji
Foley & Lardner
Kyowa Kakko Kogy Co., Ltd.
Meller Mike
Naff David M.
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