U.S. patent application number 09/885932 was filed with the patent office on 2002-03-28 for phytase having a low michaelis constant for phytic acid from monascus.
Invention is credited to Anazawa, Hideharu, Kato, Yoko, Kuroyanagi, Satoshi, Nagashima, Tadashi, Sugimoto, Seiji, Yamamura, Tadanori, Yano, Keiichi.
Application Number | 20020037571 09/885932 |
Document ID | / |
Family ID | 17253131 |
Filed Date | 2002-03-28 |
United States Patent
Application |
20020037571 |
Kind Code |
A1 |
Nagashima, Tadashi ; et
al. |
March 28, 2002 |
Phytase having a low michaelis constant for phytic acid from
monascus
Abstract
The present invention provides phytases having low Km values for
phytic acid, a process for producing the phytases by using a
microorganism having the ability to produce and accumulate the
phytases and feed containing the phytases. According to the present
invention, novel phytases 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
Inventors: |
Nagashima, Tadashi; (Aichi,
JP) ; Kuroyanagi, Satoshi; (Aichi, JP) ;
Yamamura, Tadanori; (Aichi, JP) ; Anazawa,
Hideharu; (Tokyo, JP) ; Kato, Yoko; (Tokyo,
JP) ; Sugimoto, Seiji; (Tokyo, JP) ; Yano,
Keiichi; (Tokyo, JP) |
Correspondence
Address: |
Stephen A. Bent
FOLEY & LARDNER
Washington Harbour
3000 K Street, N.W., Suite 500
Washington
DC
20007-5109
US
|
Family ID: |
17253131 |
Appl. No.: |
09/885932 |
Filed: |
June 22, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09885932 |
Jun 22, 2001 |
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09269062 |
Mar 18, 1999 |
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6261592 |
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Current U.S.
Class: |
435/195 ;
424/442 |
Current CPC
Class: |
C12N 9/16 20130101; Y10S
426/807 20130101; A23K 20/189 20160501 |
Class at
Publication: |
435/195 ;
424/442 |
International
Class: |
C12N 009/14; A23K
001/165; A23K 001/17 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 25, 1996 |
JP |
253564/1996 |
Claims
1. Phytases having Michaelis constants (referred to hereinafter as
Km) of 10 to 110 .mu.M when phytic acid is used as a substrate.
2. Phytases according to claim 1, wherein phytase I has the
following physicochemical properties: 1) Km: 27 .mu.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.degree. C.; 5) temperature stability: stable up to
35.degree. 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-phosph- ate, 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).
3. Phytases according to claim 1, wherein phytase II has the
following physicochemical properties: 1) Km: 20 .mu.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.degree. C.; 5) temperature stability: stable up to
50.degree. 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-phosph- ate, 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).
4. Phytases according to claim 1, wherein phytase III has the
following physicochemical properties: 1) Km: 107 .mu.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.degree. C.; 5) temperature stability: stable up to
60.degree. 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-phosph- ate, D-myo-inositol-1,4-diphosphate, and
adenosine triphosphate as the substrate; 7) molecular weight: about
140 kDa (gel filtration method); 8) isoelectric point: pI 5.2
(chromatofocusing method); and 9) N-terminal amino acid sequence:
shown in SEQ ID NO:1.
5. Phytase according to claim 1, 2, 3 or 4 wherein the phytases are
phytases derived from a microorganism belonging to the genus
Monascus.
6. Phytases according to claim 5 wherein the microorganism
belonging to the genus Monascus is Monascus anka IFO30873.
7. A process for producing phytases which comprises culturing a
microorganism having the ability to form and accumulate phytases
having Michaelis constants of 10 to 110 .mu.M for phytic acid as a
substrate until the phytases are formed and accumulated, and
recovering the phytases from the culture.
8. The process according to claim 7, wherein phytase I is a phytase
having the following physicochemical properties: 1) Km: 27 .mu.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.degree. C.; 5) temperature stability: stable up to
35.degree. 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).
9. A process according to claim 7, wherein phytase II is a phytase
having the following physicochemical properties: 1) Km: 20 .mu.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.degree. C.; 5) temperature stability: stable up to
50.degree. 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).
10. A process according to claim 7, wherein phytase III is a
phytase having the following physicochemical properties: 1) Km: 107
.mu.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.degree. C.; 5) temperature stability:
stable up to 60.degree. 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); 8) isoelectric point: pI 5.2
(chromatofocusing method); and 9) N-terminal amino acid sequence
shown in SEQ ID NO:1.
11. The process according to claim 7, 8, 9 or 10 wherein the
phytase is a phytase derived from a microorganism belonging to the
genus Monascus.
12. The process according to claim 11 wherein the microorganism
belonging to the genus Monascus is Monascus anka IFO30873.
13. Animal feed comprising phytases having Michaelis constants of
10 to 110 .mu.M when using phytic acid as a substrate.
Description
TECHNICAL FIELD
[0001] 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
[0002] 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.
[0003] 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.
[0004] 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).
[0005] 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.
[0006] The Michaelis constants (Km) of known phytases derived from
filamentous fungi are 250 .mu.M for Aspergillus ficuum (WO
91/05053) and 330 .mu.M Aspergillus oryzae (Biosci. Biotech.
Biochem., 57, 1364-1365 (1993)).
[0007] 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.
[0008] 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
[0009] 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.
[0010] 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 .mu.M when phytic acid was used as a
substrate, and they revealed the properties thereof and established
a process for producing said phytases to complete the present
invention.
[0011] That is, the present invention relates to novel phytases
having Km values of 10 to 110 .mu.M and a process for producing
said phytases.
[0012] Specific examples of the novel phytases of the invention
include 3 phytases having the following physicochemical
properties:
[0013] 1. Phytase I
[0014] 1) Km: 27 .mu.M when phytic acid is used as a substrate;
[0015] 2) optimum pH: pH 5.5;
[0016] 3) pH stability: stable in the range of pH 5.5 to 6.5;
[0017] 4) optimum temperature: 50.degree. C.;
[0018] 5) temperature stability: stable up to 35.degree. C;
[0019] 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;
[0020] 7) molecular weight: about 80 to 100 kDa (gel filtration
method); and
[0021] 8) isoelectric point: pI 5.7 (chromatofocusing method)
[0022] 2. Phytase II
[0023] 1) Km: 20 .mu.M when phytic acid is used as a substrate;
[0024] 2) optimum pH: pH 6.0;
[0025] 3) pH stability: stable in the range of pH 6.0 to 7.0;
[0026] 4) optimum temperature: 50.degree. C.;
[0027] 5) temperature stability: stable up to 50.degree. C.;
[0028] 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;
[0029] 7) molecular weight: about 120 kDa (gel filtration method);
and
[0030] 8) isoelectric point: pI 4.8 (chromatofocusing method).
[0031] 3. Phytase III
[0032] 1) Km: 107 .mu.M when phytic acid is used as a
substrate;
[0033] 2) optimum pH: pH 2.5;
[0034] 3) pH stability: stable in the range of pH 2.0 to 8.0;
[0035] 4) optimum temperature: 45.degree. C.;
[0036] 5) temperature stability: stable up to 60.degree. C.;
[0037] 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;
[0038] 7) molecular weight: about 140 kDa (gel filtration method);
and
[0039] 8) isoelectric point: pI 5.2 (chromatofocusing method);
and
[0040] 9) N-terminal amino acid sequence: shown in SEQ ID NO:
1.
[0041] The microorganisms used in the present invention may be any
microorganisms producing the novel phytases having Km values of 10
to 110 .mu.M when phytic acid is used as a substrate, and examples
are microorganisms belonging to the genus Monascus. Specifically,
Monascus anka IFO3087 can be mentioned. Further, animal cells
having the ability to produce the novel phytases, which have Km
values of 10 to 110 .mu.M when phytic acid is used as a substrate,
can also be used in the present invention.
[0042] The microorganism having the ability to produce the novel
phytase is cultured 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 novel phytase-producing
organism.
[0043] If the novel phytase-producing organism is a prokaryote such
as Escherichia coli or an eukaryote such as filamentous fungus,
yeast etc., the medium for culturing said microorganism may be a
natural or synthetic medium insofar as the medium contains a carbon
source, a nitrogen source, and inorganic salts etc. which can be
assimilated by the microorganism and in which the microorganism can
be efficiently cultured.
[0044] The carbon source may be any one which can be assimilated by
the microorganism and includes glucose, fructose, sucrose, molasses
containing such sugar, hydrocarbons such as starch, starch
hydrolysates etc., organic acids such as acetic acid, propionic
acid etc., and alcohols such as ethanol, propanol etc.
[0045] The nitrogen source includes ammonia, ammonium salts of
various inorganic and organic acids, such ammonium chloride,
ammonium sulfate, ammonium acetate, ammonium phosphate etc. and
other nitrogenous compounds, as well as peptone, meat extract,
yeast extract, corn steep liquor, casein hydrolysates, soybean
cake, soybean cake hydrolysates, and a wide variety of
microorganisms obtained by fermentation and digested materials
thereof.
[0046] Inorganic materials include potassium dihydrogen phosphate,
dipotassium hydrogen phosphate, magnesium phosphate, magnesium
sulfate, sodium chloride, ferrous sulfate, manganese sulfate,
copper sulfate, calcium carbonate etc.
[0047] Further, a medium containing wheat bran, rice bran etc. as
the carbon, nitrogen and inorganic sources supplemented with
suitable salts can be used as a medium in culturing filamentous
fungi.
[0048] Culture is conducted under aerobic conditions in shaking
culture or in submerged spinner culture under aeration. The culture
temperature is preferably 15 to 40.degree. C., and the culturing
period is usually 16 to 96 hours. The pH during culture is
maintained in the range of 3.0 to 9.0. Adjustment of the medium pH
is conducted with inorganic or organic acid, alkali solution, urea,
calcium carbonate or ammonia.
[0049] During culture, antibiotics such as ampicillin, tetracycline
etc. may be added to the medium.
[0050] If filamentous fungi are to be cultured in a medium
containing solid components such as wheat bran etc., the
filamentous fungi are inoculated, mixed sufficiently with the solid
components, and spread as a thin layer on a large number of
aluminum or stainless steel trays in a cellar and cultured under
the controlled conditions of temperature, humidity and ventilation.
Specifically, the fungi are subjected to stationary culture in a
culture chamber under 100% humidity at 25 to 35.degree. C. for 3 to
10 days.
[0051] If the novel phytase-producing organism is animal cells, the
medium for culturing the animal cells includes generally used RPMI
1640 medium, Eagle's MEM medium, and mediums containing fetal
bovine serum in the above mediums, etc.
[0052] Culture is conducted under such conditions as in the
presence of 5% CO.sub.2 etc. The culture temperature is preferably
35 to 37.degree. C., and the culturing period is usually 3 to 7
days.
[0053] During culture, antibiotics such as kanamycin, penicillin
etc. may be added to the medium.
[0054] To isolate and purify the novel phytase from the culture of
the novel phytase-producing organism, conventional enzyme isolation
and purification methods may be used.
[0055] For example, if the novel phytase is accumulated in cells of
the novel phytase-producing organism, the cells are collected from
the culture by centrifugation, then washed and disrupted by a
sonicator, a French press, a Manton-Gaulin homogenizer, a dynomill
or the like whereby a cell-free extract is obtained. A supernatant
obtained by centrifuging the cell-free extract is subjected to
salting-out with e.g. sulfate ammonium, desalting, precipitation
with an organic solvent, anion-exchange chromatography on resin
such as diethylaminoethyl (DEAE)-Sepharose and DIAION HPA-75
(Mitsubishi Chemical Industries Ltd.), cation-exchange
chromatography on resin such as S-Sepharose FF (Pharmacia),
hydrophobic chromatography on resin such as butyl Sepharose and
phenyl Sepharose, gel filtration on molecular sieves,
chromatofocusing and electrophoresis such as isoelectric focusing,
whereby a purified enzyme preparation can be obtained.
[0056] Analysis of the structure of the purified enzyme preparation
can be effected by techniques generally used in protein chemistry,
for example techniques described in "Protein Structural Analysis
for Gene Cloning" authored by Hisashi Hirano and published by Tokyo
Kagaku Dojin (1993).
[0057] If the novel phytase is extracellularly secreted, the
culture is subjected to e.g. centrifugation to give a soluble
fraction. If solid components such as wheat bran etc. are present
in the medium ingredients, the novel phytase is extracted with hot
water or the like and subjected to techniques such as
centrifugation to give a soluble fraction. From this soluble
fraction, a purified enzyme preparation of the novel phytase can be
obtained by the same techniques as in isolation and purification
from the supernatant of the cell-free extract as described
above.
[0058] In the present invention, the activity of the novel phytase
can be determined according to a standard activity measurement
method (see the Reference Example below).
[0059] Further, the Km value of the novel phytase can be determined
by the Lineweaver-Burk plot in which the activity of the novel
phytase, as determined by the standard activity measurement method,
is plotted at varying concentrations of the substrate.
[0060] The novel phytase of the invention can be utilized in
various steps required for converting a salt of phytate into
inositol and inorganic phosphate, for example in producing animal
feed, soybean processing, liquid feed for pigs and poultry, and
inositol or inositol monophosphate from salts of phytate.
[0061] Animal feed containing the novel phytase of the invention
can be produced by mixing said enzyme with carriers such as wheat
chaff, drying the mixture in a spraying column or a fluidized bed,
and adding osmotic pressure stabilizers such as sorbitol and
preservatives such as benzoic acid to the dried material. The
amount of the novel phytase in animal feed is 10 to 5000 U,
preferably 100 to 1000 U, per kg of the animal feed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0062] FIG. 1 shows the optimum pH of phytase I.
[0063] FIG. 2 shows the optimum pH of phytase II.
[0064] FIG. 3 shows the optimum pH of phytase III.
[0065] FIG. 4 shows the pH stability of phytase I.
[0066] FIG. 5 shows the pH stability of phytase II.
[0067] FIG. 6 shows the pH stability of phytase III.
[0068] FIG. 7 shows the optimum temperature of phytase I.
[0069] FIG. 8 shows the optimum temperature of phytase II.
[0070] FIG. 9 shows the optimum temperature of phytase III.
[0071] FIG. 10 shows the temperature stability of phytase I.
[0072] FIG. 11 shows the temperature stability of phytase II.
[0073] FIG. 12 shows the temperature stability of phytase III.
BEST MODE FOR CARRYING OUT THE INVENTION
[0074] Hereinafter, the present invention is described in detail by
reference to the Examples. However, the present invention is not
limited to the Examples.
Reference Example (Method for Measuring Phytase Activity)
[0075] In the present invention, the standard activity measurement
of the phytase was conducted in the following manner.
[0076] 0.5 ml of 0.2 M acetate buffer, pH 5.5 (or glycine-HCl
buffer, pH 2.5 in the case of examination of the acidic phosphatase
activity of decomposing phytic acid) containing 2.5 mM sodium
phytate (Sigma) was kept at 37.degree. C. for 5 minutes, and 0.5 ml
of an enzyme solution was added to initiate the reaction (finally
1.25 mM sodium phytate-0.1 M acetate buffer, pH 5.5, or 0.1 M
glycine-HCl buffer, pH 2.5). After kept at 37.degree. C. for 20
minutes, 2 ml of an enzyme reaction termination solution (i.e. a
mixture consisting of 10 mM ammonium molybdate, 5 N sulfuric acid,
and acetone at the ratio of 1:1:2) was added to terminate the
reaction, and 0.1 ml of 1 M citric acid was added and mixed with
the reaction solution. The absorbance of this solution at 380 nm
was measured by a spectrophotometer (Hitachi U-2000). 1 unit of the
phytase activity is defined as the amount of the enzyme allowing to
release of 1 .mu.mol inorganic phosphorus per minute.
Example 1 (Production of Phytases)
[0077] Into a 2000 ml Erlenmeyer flask, 200 ml of a phytase
production medium [1% sucrose, 0.2% NaNO.sub.3, 0.05%
MgSO.sub.4.7H.sub.2O, 0.05% KCl, 0.001% FeSO.sub.4.7H.sub.2O, 0.1%
corn steep liquor (C. S. L.), pH 5.5] was introduced, capped with a
silicone sponge stopper, and sterilized at 120.degree. C. for 20
minutes. Hyphae of Monascus anka IFO30873 were inoculated into it
and subjected to stationary culture for 10 days to produce
phytases. As a crude phytase solution, 980 ml of the resulting
culture was used and about 6 units of phytases were thus
obtained.
Example 2 (Purification of the Phytases)
[0078] The crude phytase solution was desalted by passing it
through an Ultrafilter (an exclusion molecular weight of 10,000,
Advantec). The resulting enzyme solution was applied to a
DEAE-Sepharose F. F. (Pharmacia) column previously equilibrated
with 20 MM MES buffer, pH 6.0 (Katayama Kagaku). After washed with
20 mM MES buffer (pH 6.0), phytases I and III were eluted with 20
mM MES buffer (pH 6.0) containing 0.05 M NaCl. Further, phytase II
was eluted with 20 mM MES buffer (pH 6.0) containing 0.1 M NaCl.
Each enzyme solution thus separated was desalted and concentrated
about 20-fold respectively with an Ultrafilter (an exclusion
molecular weight of 10,000, Advantec). Further, each enzyme was
applied to a DEAE-Sepharose F. F. (Pharmacia) column previously
equilibrated with 20 mM MES buffer (pH 6.0). After washed with 20
mM MES buffer (pH 6.0), the protein was eluted with a linear
gradient of 0 to 0.3 M NaCl whereby phytase I, II and III fractions
were obtained. Each of the enzyme solutions thus obtained was
concentrated about 20-fold with an Ultrafilter (an exclusion
molecular weight of 10,000, Advantec) and applied to a TOYO-pearl
HW-55F (Tosoh Corporation) column previously equilibrated with 20
mM MES buffer, pH 6.0 containing 0.05 M KCl. Phytases I, II and III
were eluted respectively with 20 mM MES buffer (pH 6.0) containing
0.05 M KCl.
[0079] Phytases I, II and III were purified respectively in the
steps described above.
Example 3 (Measurement of Km of Phytases I, II and III)
[0080] The purified phytases I, II and III were measured according
to the standard activity measurement method where phytic acid was
used as a substrate at a varying concentration in the range of
0.00625 to 0.8 mM. The Lineweaver-Burk reciprocal of the result was
plotted to determine Km. The Km values of phytases I, II and III
for phytic acid were 27, 20 and 107 .mu.M.
Example 4 (Physicochemical Properties of Phytases I, II and
III)
[0081] {circle over (1)} Optimum pH: The buffer in the standard
activity measurement method was replaced by 0.2 M buffer below, and
the activity at each pH was measured according to the standard
activity measurement method.
1 Glycine-HCl buffer pH 2.0 to 3.5 Acetic acid-sodium acetate
buffer pH 3.5 to 5.5 MES buffer (Good buffer) pH 5.0 to 7.0
Tris-HCl buffer pH 7.0 to 9.0
[0082] The results are shown in FIGS. 1, 2 and 3.
[0083] Phytases I, II and III exhibited the maximum activity at pH
5.5, pH 6.0 and pH 2.5, respectively.
[0084] {circle over (2)} pH stability: After the enzyme solution
was kept at 30.degree. C. for 60 minutes in 50 mM buffer below, the
activity was measured according to the standard activity
measurement method.
2 Glycine-HCl buffer pH 2.0 to 4.0 Acetic acid-sodium acetate
buffer pH 4.0 to 5.5 MES buffer (Good buffer) pH 5.5 to 7.0
Tris-HCl buffer pH 7.0 to 9.0
[0085] The results are shown in FIGS. 4, 5 and 6.
[0086] Phytase I was stable in the range of pH 5.5 to 6.5, phytase
II in the range of pH 6.0 to 7.0, and phytase III in the range of
pH 2.0 to 8.0.
[0087] {circle over (3)} Optimum temperature: After the reaction
temperature used for measuring the enzyme activity was varied in
the range of 30 to 70.degree. C., the activity at each temperature
was measured according to the standard activity measurement
method.
[0088] The results are shown in FIGS. 7, 8 and 9.
[0089] Both phytases I and II showed the maximum activity at
50.degree. C. Phytase III showed the maximum activity at 45.degree.
C.
[0090] {circle over (4)} Temperature stability: After the enzyme
solution was kept at 4 to 70.degree. C. for 10 minutes in 20 mM MES
buffer (pH 6.0), the activity was measured according to the
standard activity measurement method.
[0091] The results are shown in FIGS. 10, 11 and 12.
[0092] Phytase I was stable up to 35.degree. C., phytase II up to
50.degree. C., and phytase III up to 60.degree. C.
[0093] {circle over (5)} Substrate specificity: The activity was
determined according to the standard activity measurement method
using each substrate prepared at 2.5 mM, and the results are shown
in Table 1.
[0094] Any of phytases I, II and III had low substrate
specificity.
3TABLE 1 Substrate Specificity of the Phytases Phytases I II III
Phytic acid 100 100 37.8 p-Nitrophenylphosphate 224 53.9 100
D-myo-inositol (1,4)-diphosphate 99.6 53.5 93.9 D-myo-inositol
2-phosphate 53.9 11.1 43.5 D-myo-inositol 1-phosphate 87.9 17.7
53.5 Glucose 6-phosphate 152 30.9 128 Fructose 6-phosphate 211 29.6
95.0 Adenosine triphosphate 223 42.0 191
[0095] {circle over (6)} Molecular weight: The molecular weight was
determined by gel filtration on TOYO-pearl HW-55F (column size
1.0.times.119 cm, 93.4 ml). A calibration curve was prepared using
RNase A (M. W. 15500), ovalbumin (M. W. 42700), albumin (M. W.
63300), and aldolase (M. W. 163000) as standards.
[0096] The molecular weights of phytases I, II and III were 80 to
100, 120 and 140 kDa, respectively.
[0097] {circle over (7)} Isoelectric point: Phytase was applied to
a Polybuffer exchanger PBE94 column (Pharmacia) previously
equilibrated with 20 mM MES buffer (pH 6.0), and then eluted with
10% Polybuffer 74-HCl (pH 4.0). The isoelectric point of the
phytase was determined by measuring the pH value of the elution
fraction with a pH meter.
[0098] The results indicated that the isoelectric points of
phytases I, II and III were pI 5.7, 4.8 and 5.2, respectively.
Example 5 (Analysis of N-terminal Amino Acid Sequence of Phytase
III)
[0099] To determine the molecular weight of phytase III by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the enzyme was
highly purified. To 12.5% multi-gel (Daiichi Kagaku K. K.), 50
.mu.l of the enzyme purified according to Example 2 was applied and
subjected to native-PAGE according to the method of B. J. Davis
[Ann. N.Y. Acad. Sci., 121, 404-427 (1964)]. After electrophoresis,
the gel was sliced into pieces of 5 mm in width and immersed in 1
ml of 20 mM MES buffer (pH 6.0) and stored at 4.degree. C. for 12
hours so that the enzyme was extracted. After the extract was
concentrated 15- to 20-fold in a rotary evaporator, a phytase
fraction was identified by the standard activity measurement method
described in the Reference Example. Under reduction with
mercaptoethanol, 10 .mu.l of the active fraction was subjected to
SDS-PAGE to give high-purity phytase III. From this result, the
molecular weight of phytase III by SDS-PAGE was determined to be
about 65 kDa.
[0100] Analysis of the N-terminal amino acid sequence of phytase
III was performed in the following manner. That is, phytase III
purified in Example 2 was subjected to SDS-PAGE under reduction
with 2-mercaptoethanol and then transferred electrically to a PVDF
membrane (ProBlott, Perkin Elmer) according to the method of P.
Matsudaira [J. B. C., 262, 10035-10038 (1987)]. The membrane having
the enzyme transferred thereto was stained with Coomassie Blue, and
its band with a molecular weight of about 65 kDa was cut out and
analyzed for its N-terminal amino acid sequence by a method
recommended by manufacturer's instructions in a gas-phase protein
sequencer (PPSQ-10, Shimadzu Corporation). The amino acid sequence
thus obtained is shown in SEQ ID NO:1. As a result of homology
examination of the amino acid sequence described herein by using a
protein data base, it was found to be a novel sequence.
INDUSTRIAL APPLICABILITY
[0101] According to the present invention, there can be provided
phytases having low Km values for phytic acid and a process for
producing the phytases by use of a microorganism having the ability
to produce and accumulate the phytases.
[0102] The phytases can be added to feed to 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.
Sequence CWU 1
1
1 1 17 PRT Monascus anka 1 Phe Gln Ser Val Ile Ser Glu Lys Gln Phe
Ser Gln Glu Phe Leu Asp 1 5 10 15 Asn
* * * * *