Phytase and gene encoding said phytase

Details Phytase and gene encoding said phytase Phytase and gene encoding said phytase

2000-04-05

2001-10-30

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S252300, C435S254110, C435S254300, C435S320100, C536S023200

Reexamination Certificate

active

06309870

ABSTRACT:

TECHNICAL FIELD
The present invention relates to an inexpensive phytase with a low Michaelis constant (hereinafter abbreviated to Km) for phytic acid, which degrades phytic acid as an anti-trophic factor contained in feed, thereby improving the nutritive value of the feed and enabling an efficient utilization of phosphoric acid released by the degradation; and to a gene coding for the phytase.
BACKGROUND ART
Phosphorus is an essential element for all organisms. Plant-derived feeds used for the production of domestic animals contain phosphorus, 50 to 70% of which is present as phytic acid. Phytic acid is a major storage substance of phosphoric acid, existing in a large amount in plant seeds. However, phytic acid is excreted without digestion and absorption in the digestive organs in single-stomach animals such as pigs, chickens, etc. That is, phytic acid is a storage substance of phosphoric acid, but its phosphorus is not utilized at all. Accordingly, phosphoric acid is added to feed for single-stomach animals for the purpose of growth promotion.
Addition of phosphoric acid to the feed leads to an increase in the amount of phosphorus in excrement. In recent years, excrement from domestic animals increase considerably as the production of domestic animals increases more and more, whereby an environmental problem is now caused in the world. In particular, phosphorus contained in excrement is mentioned as a factor causing the phenomenon of nutrition enrichment in lakes and marshes, so the amount of phosphorus in excrement comes to be regulated, and there arises necessity for countermeasure.
In addition to the problem of excreted phosphorus, phytic acid chelates divalent metals important as a nutritive source, such as magnesium, calcium, zinc and iron, thereby making its absorption into animals difficult and reducing the nutritive value of feed. Accordingly, phytic acid is regarded as an anti-trophic factor.
From the foregoing, it has been examined to decrease the amount of phosphorus in excrement by treating the feed with a phytase known widely as an enzyme capable of hydrolyzing a salt of phytic acid into inositol and inorganic phosphoric acid in order to utilize phosphoric acid released from phytic acid in place of phosphoric acid conventionally added in feed, and it has also been examined to improve the nutritive value of the feed by decomposing phytic acid as an anti-trophic factor [U.S. Pat. No. 3,297,548 (1967); J. Nutrition, 101, 1289-1294 (1971)].
Known as phytase-producing microorganisms are bacteria such as
Bacillus subtilis
and Pseudomonas, yeasts such as
Saccharomyces cerevisiae
, and filamentous fungi such as
Aspergillus terreus, Aspergillus ficcum
and
Aspergillus awamori.
For phytase derived from
Aspergillus ficcum
, 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).
For phytase derived from
Aspergillus awamori
, its nucleotide sequence and amino acid sequence are described in Gene, 133, 55-62 (1993).
Michaelis constants (Km) for phytases known so far are 0.57 mM for wheat bran-derived phytase [Agr. Biol. Chem., 26, 794-803 (1962)], 0.17 mM for rice bran-derived phytase [Agr. Biol. Chem., 53, 1475-1483 (1898)], 117 &mgr;M for maize (
Zea mays
)-derived phytase, 250 &mgr;M for
Aspergillus ficcum
-derived phytase (WO 91/05053), 330 &mgr;M for
Aspergillus oryzae
-derived phytase, 150 &mgr;M for
Bacillus subtilis
-derived phytase, 500 &mgr;M for
Bacillus natto
-derived phytase, and 130 &mgr;M for
Escherichia coli
-derived phytase.
To demonstrate the performance of the enzyme, the concentration of a substrate is necessary to be higher than Km, and if an enzyme with low Km and an enzyme with high Km have the same maximum reaction rate (Vmax), the enzyme with low Km does not decrease a reaction rate even at a lower substrate concentration as compared with the enzyme with high Km.
That is, when compared with the enzyme with high Km, the enzyme with low Km is advantageous in that a sufficient degradation rate can be achieved even at a lower substrate concentration, thereby minimizing the amount of the remaining substrate.
Accordingly, there is a demand for an inexpensive phytase with a low Km value for phytic acid, which phytase degrades phytic acid being an anti-trophic factor contained in feed, thereby improving the nutritive value of the feed and simultaneously achieving an efficient utilization of phosphoric acid released by the degradation.
DESCRIPTION OF THE INVENTION
The present invention relates to a phytase (hereinafter referred to as “novel phytase”) having a Michaelis constant of 10 to 30 &mgr;M when using phytic acid as the substrate, DNA coding for the phytase, recombinant DNA having the DNA introduced thereinto, a transformant carrying the recombinant DNA, a process for preparing a phytase by use of the transformant, and an animal feed containing the phytase.
The present invention will be described in detail.
A specific example of the novel phytase of the present invention includes: a phytase with the following physicochemical properties:
(1) Km value: 10 to 30 &mgr;M;
(2) molecular weight (by SDS-PAGE): about 60 kDa after treatment with endoglycosidase H;
(3) optimum pH: pH 5.0 to 6.5;
(4) optimum temperature: 45 to 65° C. showing maximum activity;
(5) substrate specificity: acting on the substrates, phytic acid, p-nitrophenylphosphate, D-glucose 6-phosphate, fructose 6-phosphate, D-myo-inositol 1,4,5-triphosphate, glycerol phosphate, and adenosine triphosphate; and
(6) isoelectric focusing: pI 4.7 to 5.4.; or a phytase protein having the amino acid sequence shown in SEQ ID NO:1 or 2.
Furthermore, the present invention encompasses a novel phytase having an amino acid sequence (for example, shown in SEQ ID NO:3) in which a secretory signal sequence has been linked to the novel phytase described above.
The present invention further includes a phytase having an amino acid sequence comprising substitutions, deletions or additions of one or more amino acids relative to the amino acid sequence shown in SEQ ID No: 1, 2 or 3; having a homology of 40% or more to the amino acid sequence shown in SEQ ID NO:1, 2 or 3; and having a Michaelis constant (Km) of 10 to 30 &mgr;M when using phytic acid as the substrate. The phytase has preferably a homology of 60% or more, more preferably 80% or more.
The substitution, deletion or addition of amino acids can be carried out according to methods described in Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Proc. Natl. Acad. Sci., USA, 81, 5662 (1984), Science, 224, 1431 (1984), PCT WO85/00817 (1985), Nature, 316, 601 (1985), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Current Protocols in Molecular Biology, chapter eight “Mutagenesis of Cloned DNA”, John Wiley & Sons, Inc. (1989), etc.
The novel phytase can also be obtained from any microorganisms having the ability to produce it. Among them, preferable examples are microorganisms belonging to the genus Aspergillus and having the ability to produce the novel phytase. More preferable examples include
Aspergillus niger
strain SK57 (FERM BP-5473) or mutants or derived strains thereof.
Aspergillus niger
strain SK92 (FERM BP-5481) is included in mutants derived from
Aspergillus niger
strain SK57.
The gene (hereinafter referred to as “novel phytase gene”) coding for the novel phytase of the present invention may be any gene coding for the novel phytase: for example, a gene coding for a phytase having the amino acid sequence shown in SEQ ID NO:1, 2 or 3; or a gene coding for a phytase which has an amino acid sequence where in the amino acid sequence shown in SEQ ID NO:1, 2 or 3, one or more amino acids have been substituted, deleted or added and which has a Michaelis constant (Km) of 10 to 30 &mgr;M when using phytic acid as the substrate. The gene may contain introns in the DNA sequence. Specifically, the gene of the present invention includes DNA shown

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