Phytase from bacillus subtilis, gene encoding said phytase,...

Details Phytase from bacillus subtilis, gene encoding said phytase,... Phytase from bacillus subtilis, gene encoding said phytase,...

2002-09-20

2003-10-28

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

Reexamination Certificate

active

06638746

ABSTRACT:

The present invention relates to phytase, nucleic acids encoding phytase as well as methods for the production of phytase and its use.
BACKGROUND OF THE INVENTION
Phosphorous is an essential element for growth. A substantial amount of the phosphorous found in many foods and animal feeds is present in the form of phosphate which is covalently bound in a molecule known as phytate (myo-inositol hexakisphosphate). Since phytate itself is poorly digested and phosphate is to a large extent absorbed in the small intestine of an animal, phosphate sequestered in phytate and not made available to an animal in the small intestine is not absorbed, passes through the digestive tract and is excreted. This leads to an increased ecological phosphorus burden to land and water. In addition, since phytate chelates several essential minerals and prevents or inhibits their absorption in the digestive tract, phytate decreases the nutritional value of food and animal feeds.
Another problem associated with poor phytate digestability is that inorganic phosphates need to be added to animal feeds, thereby increasing their costs.
Phytate is converted by enzymes known as phytases which catalyse the hydrolysis of phytate to inositol and inorganic phosphate. Phytase is found in wheat bran and plant seeds and is known to be produced by various micro-organisms including yeast, fungi and bacteria.
Among known fungal phytases,
Aspergillus terreus
phytase was purified to homogeneity by Yamada et al. (Agr. Biol. Chem., 32 (10) (1968), 1275-1282) and shown to have a pH optimum of pH 4.5, a temperature optimum of about 70° C. at pH 4.5 and a thermal stability over a temperature range from 30 to 60° C. at pH 4.5. However, said enzyme was shown to be completely inactive at neutral pH values, particularly at pH 7.0.
In addition, the
Aspergillus ficuum
phytase isolated and characterised by H. J. Ullah and D. M. Gibson (Preparative Biochemistry, 17 (1) (1987), 63-91) was shown to have two pH optima, one at 2.2 and the other at 5.0-5.5, a temperature optimum of 58° C. at pH 5.0 and a thermal stability up to 68° C. at pH 5.0. However, as is the case with
Aspergillus terreus
phytase,
Aspergillus ficuum
phytase was shown to be inactive at pH 7.0.
DNA sequences encoding phytases from
Aspergillus terreus
(EP 684 313) and
Aspergillus ficuum
(EP 420 358) as well as
Aspergillus niger
var.
awamori
(Piddington et al., (1993) Gene, 133, 55-62) have been characterised and recombinantly expressed.
Phytases are also known from bacterial sources such as
Bacillus subtilis
(V. K. Powar and V. Jagannathan, (1982) J. Bacteriology, 151 (3), 1102-1108) and
Bacillus subtilis
(natto) (M. Shimizu, (1992) Biosci. Biotech. Biochem., 56 (8), 1266-1269 and Japanese Patent Application 6-38745).
Bacillus subtilis
(natto) phytase described by Shimizu (supra) was purified to homogeneity by SDS-PAGE and was shown to have a molecular weight of between 36 and 38 kD. This enzyme was shown to have a pH optimum between pH 6.0 and 6.5 when measured in an assay solution at 37° C. comprising 0.1 M maleic acid, 2 mM CaCl
2
and 1.6 mM sodium phytate and a pH optimum of pH 7.0 when assayed in a solution comprising 0.1 M Tris-HCl buffer, 2 mM CaCl
2
and 1.6 mM sodium phytate at 37° C. The temperature optimum for this phytase was shown to be 60° C. and the enzyme is stable up to 50° C. when incubated in the above mentioned assay solution containing Tris-HCl buffer for 15 min. The specific activity of this purified
Bacillus subtilis
(natto) phytase in said Tris-HCl containing solution was reported as 8.7 U/mg protein. One unit of phytase was defined as the amount of enzyme required to liberate one &mgr;mol of Pi per minute under tha assay conditions. This definition is used throughout.
Powar et al. (supra) described the isolation of a phytate specific phosphatase preparation from
Bacillus subtilis
which has a molecular weight of 36.5 kD. This enzyme preparation, which was purified by SDS-PAGE and found to comprise two phytase isozymes, was shown to have a pH optimum between 7.0 and 7.5 when measured in an assay solution comprising 0.1 M Tris-HCl buffer, 0.5 mM CaCl
2
and 0.34 mM sodium phytate at 30° C. This phytase isozyme mixture exhibited a maximum activity at a temperature of 60° C. and was stable up to a temperature of 70° C. The specific activity of the purified enzyme was reported as 8.5 to 9.0 U/mg protein when measured in the above assay solution. In addition, it was reported by Powar et al (supra) that the purified isozyme mixture contained proteolytic activity which resulted in the loss of activity.
The amino acid sequence of Bacillus phytase as well as nucleic acids which encode Bacillus phytases are not known to date.
The idea of supplementing foods and animal feed with naturally occurring or recombinant phytases in order to enzymatically convert phytate to digestible phosphate during food and animal feed processing has been described. JP-A-6-38745 describes the use of purified naturally occurring
Bacillus subtilis
(natto) phytase for use in processing feeds and foods. In addition, EP 420 358 and EP 684 313 describe the use of Aspergillus phytase in animal feeds.
Furthermore, it has also been suggested to add phytase to animal feeds which have already been processed in order to allow the enzymatic action of said phytases to take place in the digestive tract of the animal.
However, the above mentioned Aspergillus phytases are either inactive or lose a substantial amount of their activity at the temperature and/or pH at which foods or animal feeds are processed (generally 65 to 95° C., pH 5.5 to 7.5) and at the pH of the small intestine of monogastric animals (generally 37-41° C., pH 5.5 to 7.5).
Furthermore, the specific activity, and therefore the relative activity, of the above mentioned Bacillus phytases is very low under the above conditions.
SUMMARY OF THE INVENTION
Due to the difference in the temperatures and/or pH used during processing of foodstuffs and in the digestive tract of animals, it is desirable to have available a phytase which has a high specific activity as well as a high relative activity both at the processing temperature and/or pH of foods and animal feeds and at the temperature and/or pH in the digestive tract of animals in order to both maximise the effects of phytase during food and feed processing, during digestion within the digestive tract and to reduce the phosphorous burden to the environment resulting from digestion of phytate containing animal feedstuffs.
Moreover, a method for the production of large quantities of phytase which fulfils the above criteria is also desirable for the economic production of said foods and animal feeds.
An object of the present invention is to provide phytase with a high specific activity which is capable of functioning with a high relative activity during the processing of foods and animal feeds and/or is capable of functioning with high relative activity in the digestive tract of farmed animals.
A further object of the present invention is to provide nucleic acid molecules which encode phytase of the present invention.
A further object of the present invention is to provide methods for the production of said phytase as well as means for delivering said phytase to said animals.
Other objects of the present invention will become apparent from the following detailed specification.
Subject matter of the invention is phytase or a functional derivative thereof, characterised in that said phytase has a specific activity of at least 20 U/mg protein, wherein said specific activity is determined by incubating said phytase in a solution containing 100 mM Tris-HCl, pH 7.5, 1 mM CaCl
2
, and 1.6 mM sodium phytate at 37° C. for 30 minutes. Preferably, the phytase of the present invention has a specific activity of at least 29 U/mg protein, more preferably at least 80 U/mg protein, and most preferably at least 88 U/mg protein when assayed under the above conditions.
According to a preferred embodiment, said phytase has a pH optimum of at least pH 6.5, wherein said pH optimum i

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