Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Stablizing an enzyme by forming a mixture – an adduct or a...
Reexamination Certificate
1999-10-01
2003-08-26
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Stablizing an enzyme by forming a mixture, an adduct or a...
C424S094600, C424S442000, C426S061000, C426S807000, C435S177000, C435S187000, C435S195000
Reexamination Certificate
active
06610519
ABSTRACT:
FIELD OF INVENTION
The present invention relates to solid phytase compositions which have been stabilized with a lactic acid source such as Corn Steep Liquor (CSL), and methods of producing the same.
BACKGROUND OF THE INVENTION
The addition of phytase to animal feed to eliminate the anti-nutritional effects of phytic acid is well-described, see e.g. WO 98/28408 and WO 98/28409.
The stabilization of liquid phytase formulations with urea, glycerol or sorbitol is disclosed in WO 93/16175.
Salt-stabilized solid phytase compositions are disclosed in EP 0 758 018 A1.
Plant seeds, cereal grains and legumes are usual components of animal feed. Some of those seeds contain phytic acid, and often also endogenous phytase enzymes.
According to investigations performed by the applicant, endogenous phytase activity in animal feed is at a very low level of around 0.5 units/g.
According to e.g. the two above first-cited WO-references, when supplementary phytase has been added to feed, the phytase activity in the feed is in the range of 0.01-20 units/g.
SUMMARY OF THE INVENTION
The present invention relates to solid phytase compositions which comprise (a) an enzyme having phytase activity; and (b) a lactic acid source, wherein the phytase activity of the composition is above 20 units/g.
DETAILED DESCRIPTION OF THE INVENTION
In the present context, the expression “enzyme (or polypeptide) having phytase activity” or “phytase” includes any enzyme capable of effecting the liberation of inorganic phosphate from phytic acid or from any salt thereof (phytates).
Phytic acid is myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate (or for short myo-inositol hexakisphosphate). In what follows, unless otherwise indicated, the terms “phytic acid” and “phytate,” are used synonymously or at random.
In the present context, the term “units” means units of enzyme, in particular phytase, activity. Any method for determining phytase activity can be used.
In a preferred embodiment, one unit of phytase activity is defined as the amount of enzyme that liberates 1 micro mole inorganic ortho-phosphate per min. under the following conditions: A pH which is within the range of +/−1 pH unit from the optimum pH of the actual enzyme; a temperature which is within the range of +/−20° C. from the optimum temperature of the actual enzyme; using as a substrate phytic acid or any salt thereof in a suitable concentration.
Preferably, the substrate is dodeca-sodium phytate in a concentration of 0.005 mole/l.
Preferably, the pH is within the range of +/−0.5 pH unit from the optimum pH; more preferably the pH is the optimum pH.
Preferably, the temperature is within the range of +/−10° C. from the optimum temperature; more preferably the temperature is the optimum temperature.
Preferably, the optimum pH and optimum temperature refers to the use of sodium phytate as a substrate.
In another preferred embodiment, the phytase activity is determined in the unit of FYT, one FYT being the amount of enzyme that liberates 1 micro mole inorganic ortho-phosphate per min. under the following conditions: pH 5.5; temperature 37° C.; substrate: sodium phytate (C
6
H
6
O
24
P
6
Na
12
)in a concentration of 0.0050 mole/l.
In a further preferred embodiment, the phytase activity is measured using the FTU assay.
The FYT- and FTU-assays are described in more detail in the experimental part.
In preferred embodiments, the phytase activity of the solid composition of the invention is above 25, 50, 100, 250, 500, 750 or even above 1000 units/g.
Optionally, the phytase activity of the solid composition is below 100,000 units/g, more preferably below 75,000 units/g, even more preferably below 50,000 units/g, or below 40,000 units/g, or below 25,000 units/g, or even below 10,000 units/g, mostly preferred below 5,000 units/g.
Preferred ranges of phytase activity are 25-100,000, 25-75,000, 35-50,000, or 50-40,000 units/g; more preferably 100-25.000 units/g; even more preferably 500-10.000 units/g; mostly preferred 1000-5000 units/g.
In the present context, any enzyme having phytase activity can be used.
Phytases have been derived from plants as well as from microorganisms. 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).
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, primarily belonging to the fungal phylum 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.
Phytases derived from fungi of the phylum Basidiomycota are disclosed in WO 98/28409 and WO 98/28408.
Modified phytases or phytase variants are obtainable by methods known in the art, in particular by the methods disclosed in EP 0897010, EP 0897985, PCT/DK99/00153 and PCT/DK99/00154. The phytases disclosed in either of these four patent applications can also be used in the compositions of the present invention.
A solid or dry composition is a particulate material comprising, preferably consisting essentially of, or consisting of, freely flowing particles of a size ranging from (&mgr;m) 0.01, or from 1.0, or preferably from around 1-to 1000, or to 1200, or to 1500, or even up to 2000 (&mgr;m).
Preferably, a solid or dry phytase composition is such composition which can be prepared from liquid phytase concentrates e.g by spray drying, spray cooling (prilling), or any type of granulation.
For spray drying, no further components need to be added to the liquid phytase concentrate.
For spray cooling, a meltable component—such as palm oil (and/or another meltable vegetable oil or fat), hydrogenated palm oil (and/or another hydrogenated vegetable oil), tallow, hydrogenated tallow or a wax functions as a matrix. The phytase and other ingredients, if any, are introduced into the melted, meltable component, and the melt is then allowed to solidify under particle-forming conditions, typically in a spray drying tower.
For many uses, however, including the use in animal feed, granulates are usually preferred for a number of reasons. One reason being that they may readily be mixed with feed components, or more preferably, form a component of a pre-mix which contains other desired feed additives such as vitamins and minerals.
The particle size of the enzyme granulates preferably is compatible with that of the other components of the mixture. This provides a safe and convenient mean of incorporating enzymes into, e.g., animal feed.
The size of a particle may be regarded as the greatest linear dimension of the particle; thus, in the case of, e.g., a substantially spherical parti
Henriksen Lotte Rugholm
Marcussen Erik
Lambiris Elias J.
Naff David M.
Novozymes A/S
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