U.S. patent application number 11/610725 was filed with the patent office on 2007-07-12 for polynucleotides encoding phytase polypeptides.
This patent application is currently assigned to FORSKNINGSCENTER RISO. Invention is credited to Katja Salomon Johansen, Soren Rasmussen, Mikael Blom Sorensen.
Application Number | 20070163009 11/610725 |
Document ID | / |
Family ID | 8159472 |
Filed Date | 2007-07-12 |
United States Patent
Application |
20070163009 |
Kind Code |
A1 |
Rasmussen; Soren ; et
al. |
July 12, 2007 |
POLYNUCLEOTIDES ENCODING PHYTASE POLYPEPTIDES
Abstract
The present invention relates to barley isolated polypeptides
having phytase activity, recombinant DNA sequences encoding such
polypeptides, methods of producing such polypeptides and the use of
said polypeptides in transgenic plants. The invention disclose
polypeptides having affinity for the substrate phytate (1,2,3,4,5,6
myo-inositol-hexakisphosphate, phytic acid), comprising an amino
acid sequences as described by the invention. Furthermore, the
invention discloses DNA fragments encoding said polypeptides, and
cDNA fragments encoding said polypeptides. The polypeptides of the
invention may for example be used as an additive in animal feeds,
an additive in food for human consumption or to extract proteins
from rice bran. The invention also concerns a transgenic plant or
part thereof, wherein said plant or part thereof have been
genetically modified to comprise a polypeptide as defined in by the
invention.
Inventors: |
Rasmussen; Soren; (Roskilde,
DK) ; Sorensen; Mikael Blom; (Copenhagen, DK)
; Johansen; Katja Salomon; (Gentofte, DK) |
Correspondence
Address: |
CHRISTENSEN, O'CONNOR, JOHNSON, KINDNESS, PLLC
1420 FIFTH AVENUE
SUITE 2800
SEATTLE
WA
98101-2347
US
|
Assignee: |
FORSKNINGSCENTER RISO
Frederiksborgvej 399
Roskilde
DK
DK-4000
PLANT BIOSCIENCE LIMITED
NORWICH RESEARCH PARK COLNEY LANE, NORWICH
NORFOLK
GB
NR47
|
Family ID: |
8159472 |
Appl. No.: |
11/610725 |
Filed: |
December 14, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10275311 |
May 5, 2003 |
7186817 |
|
|
PCT/DK01/00314 |
May 4, 2001 |
|
|
|
11610725 |
Dec 14, 2006 |
|
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|
Current U.S.
Class: |
800/320 ;
435/196; 435/419; 435/468; 536/23.2; 800/320.1; 800/320.2;
800/320.3 |
Current CPC
Class: |
C12N 15/8243 20130101;
A23V 2300/21 20130101; A23K 40/10 20160501; A23L 33/18 20160801;
A23V 2002/00 20130101; C12N 9/16 20130101; A23K 20/189 20160501;
A23V 2002/00 20130101 |
Class at
Publication: |
800/320 ;
435/196; 536/023.2; 435/419; 435/468; 800/320.1; 800/320.2;
800/320.3 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C07H 21/04 20060101 C07H021/04; C12N 9/16 20060101
C12N009/16; C12N 5/04 20060101 C12N005/04 |
Foreign Application Data
Date |
Code |
Application Number |
May 4, 2000 |
DK |
PA 2000 00741 |
Claims
1. An isolated polypeptide having affinity for the substrate
phytate (1,2,3,4,5,6 myo-inositol-hexakisphosphate, phytic acid),
comprising an amino acid sequence as given in SEQ ID NO:1 or an
equivalent thereof, having a sequence identity with SEQ ID NO:1 of
at least 70% and at least one or more of the amino acid sequences
as given in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ
ID NO:7, SEQ ID NO:9, or SEQ ID NO:12, or an equivalent thereof
having a sequence identify with SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:12 of at
least 70%.
2. A polypeptide according to claim 1, capable of binding a
monoclonal antibody raised against a polypeptide having the
sequence of SEQ ID NO:1.
3. A polypeptide of claim 1, comprising an N-terininal amino acid
sequence as given in SEQ ID NO:1 or an equivalent thereof, having a
sequence identity of with SEQ ID NO:1 of at least 70%.
4. A polypeptide of claim 1, having a denatured molecular weight of
between 50-75 kDa, preferably 53-70 kDa, more preferably 55-68 kDa
as determined by SDS-PAGE.
5. A polypeptide of claim 1, having a native molecular weight of
35-55 kDa as determined by gelfiltration.
6. A polypeptide of claim 1, having a substrate affinity of
0.05-0.50 mM.
7. A polypeptide of claim 1, having a V.sub.max of 600-1000
U/mg.
8. A polypeptide of claim 1, having an isoelectric point of between
5.0-8.0.
9. A polypeptide of claim 1, having a pH optimum of between 4 and
8, preferably between 5-7.
10. A polypeptide of claim 1, wherein the activity is optimal at a
temperature between 30.degree.-65.degree. C., preferably between
35.degree.-60.degree. C., and more preferably between
40.degree.-55.degree. C.
11. A polypeptide according to claim 1, exhibiting 6-phytase
activity.
12. A DNA fragment encoding a polypeptide as defined in claim
1.
13. A cDNA fragment encoding a polypeptide as defined in claim
1.
14. A cDNA fragment encoding a polypeptide according to claim 13
having an amino acid sequence as given in SEQ ID NO:8, or an
equivalent thereof having a sequence identity with SEQ ID NO:8 of
at least 70%, such as at least 80%, for example at least 85%, such
as at least 90%, for example at least 95%.
15. A DNA fragment encoding a polypeptide according to claim 12
having an amino acid sequence as given in SEQ ID NO: 10 or an
equivalent thereof having a sequence identity with SEQ ID NO:10 of
at least 80%, such as 90%.
16. A DNA fragment encoding a polypeptide according to claim 12
having an amino acid sequence as given in SEQ ID NO:11 or an
equivalent thereof having a sequence identity with SEQ ID NO:11 of
at least 80%, such as 90%.
17. An expression cassette comprising a DNA fragment as defined in
claim 12.
18. A cell which is capable of expressing a polypeptide and which
is transformed with an expression cassette as defined in claim
17.
19. A method of producing a polypeptide having affinity for the
substrate phytate (1,2,3,4,5,6 myo-inositol-hexakisphosphate,
phytic acid) from a grass or cereal comprising the steps of: short
time extraction of the polypeptide, obtaining an extract,
subjecting the extract to purification steps, purifying the
polypeptide from said extract, obtaining the polypeptide.
20. A method according to claim 19 additionally comprising the
steps of: exposing the polypeptide to epitope specific antibodies
raised against said polypeptide, assessing the specificity by
Western blotting.
21. A method of producing a recombinant polypeptide having affinity
for the substrate phytate (1,2,3,4,5,6
myo-inositol-hexakisphosphate, phytic acid), comprising culturing a
cell as defined in claim 18, in a suitable culture medium under
conditions allowing expression of the polypeptide, and recovering
the polypeptide from the culture.
22. A product obtained by the method as defined in claim 19.
23. A product obtained by the method as defined in claim 20.
24. Use of a polypeptide as defined in claim 1.
25. The use according to claim 24 as an additive in animal
feeds.
26. The use according to claim 24 as an additive in food for human
consumption.
27. The use according to claim 24 as an industrial processing
enzyme.
28. The use according to claim 24 for the extraction of protein
from rice bran.
29. The use according to claim 24 in a transgenic plant or part
thereof.
30. A transgenic plant or part thereof, wherein said plant or part
thereof have been genetically modified to comprise a polypeptide as
defined in claim 1.
31. A transgenic plant according to claim 30, being a plant
selected from wheat, barley, rye, spelt, oat, rice or maize.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to isolated polypeptides
having phytase activity, recombinant DNA sequences encoding such
polypeptides, methods of producing such polypeptides and the use of
said polypeptides in transgenic plants.
BACKGROUND OF THE INVENTION
[0002] Phytate is the major storage compound of phosphate in plant
seeds and binds up to 80% of the total phosphate content in cereal
grains (Eeckhout and Depaepe, 1994). It consists of a six-carbon
ring with six phosphate groups attached. The negative charge is
balanced by cations of magnesium, calcium among others. Together
they form large crystals that are stored within the aleurone embryo
or the endosperm of the seed.
[0003] Monogastric animals generally are not able to degrade the
phytate present in the feed and the phosphate compound is thus,
through the manure, administered to the environment. This leads to
eutrophication of lakes, streams and the coastal sea, which results
in increased growth of algae and in the end to sub-oxygen regimes
and the death of aquatic life. For people with unbalanced diets as
seen in many undeveloped countries the insufficient digestion of
phytate is a severe nutritional problem, particularly because it is
sequestering zinc and iron from uptake. The formation of insoluble
aggregates of phytate with important minerals as zinc and iron as
well as with proteins leads to poor digestibility of all the
agents.
[0004] The scientific interest in phytate and its metabolic enzymes
goes back more than a century, though it probably received the most
attention from the general public in the early seventies With the
reintroduction of vegetable diets and wholemeal bread the
degradation of phytate became a problem for human nutrition in the
western world. Today phytate related concerns in the western world
involves the negative effects on the environment caused by the
intensive production of fish, pigs and poultry.
[0005] Phytate is the trivial name for the mixed salt of
1,2,3,4,5,6 myo-inositol-hexakisphosphate or phytic acid
(InsP.sub.6). Myo-inositol is synthesised from D-glucose via three
enzymatic steps, a) hexokinase (EC 2.7.1.1), b) 1L-myo-inositol
1-phosphate synthase (EC 5.5.1.4) and c) myo-inositol 1-phosphate
phosphatase (Loewus and Murthy, 2000).
[0006] Phytate (InsP.sub.6) is believed to function as an effective
storage compound in the seed of both phosphate and essential
cations, especially potassium and magnesium. The inositol moiety,
the phosphate groups as well as the chelated cations are believed
to be utilised by the growing seedling.
[0007] Where lnsP.sub.6 in the plant cells have been assigned a
pure storage function and as a precursor of the lower lnsP, recent
reports have shown InsP.sub.6 to act as a signalling molecule
(Voglmaier et al., 1992), (Larsson et al., 1997) in animal systems,
in yeast (York et al., 1999) and in plants (Munnik et al., 1998),
(Muir and Sanders, 1997)
[0008] Phytases are a group of phosphatases that catalysis the
stepwise removal of orthophosphate from phytate. Phytase enzymes
are classified into two groups according to the initial position of
hydrolysis. All the fungal phytases investigated as well as the
novel phytases from Bacillus subtilis and B. amyloliquefaciens
(Kerovuo et al., 1998 & 2000; Kim et al., 1998) initiate the
hydrolysis of phytate at position 3 (EC 3.1.3.8) and catalyse the
reaction: myo-Inositol
hexakisphosphate+H.sub.2O.fwdarw.D-myo-Inositol-1,2,4,5,6-pentakisphospha-
te+orthophosphate
[0009] (The Bacillus enzyme is stated in the EMBL accession as
3-phytase but it has not been published elsewhere). The plant
phytases as well as the enzyme from E. coli are 6-phytases (EC
3.1.3.26) and catalyse the reaction: myo-Inositol
hexakisphosphate+H.sub.2O.fwdarw.1L-myo-Inositol-1,2,3,4,5-pentakisphosph-
ate+orthophosphate
[0010] In this instance the L-configuration is used and the removed
phospho-group in the latter reaction scheme is situated at position
4 when the D-configuration is assigned.
[0011] Most phytases identified are enzymes that accept a broad
range of substrates and as such, phytases are a rather loosely
defined subclass of phosphatases.
[0012] Numerous phytase enzymes have been characterised from
fungal, bacterial, animal and plant sources (Dvorakova, 1998).
However, microbiel phytase enzymes are by far the best known. These
include Aspergillus niger PhyB (Ehrlich et al., 1993), A. fumigatus
(Ullah and Dischinger, 1993), A. niger PhyA (van Hartingsveldt et
al., 1993), A. niger w. awamori (Piddington et al., 1993), A.
terreus (Mitchell et al., 1997), A. ficcum (Ullah and Dischinger,
1993), Emericella nidulans (Aspergillus nidulans) (Mitchell et al.,
1997), the heat tolerant Talaromyces thermophilus (Pasamontes et
al., 1997), E. coli (Jia et al., 1998), Bacillus sp. (Kim et al.,
1998) and Bacillus subtilis (Kerovuo et al., 1998).
[0013] Among plants phytase activity from wheat, rye, spelt, oat,
rice and maize have all been subjected to purification and
characterisation procedures. Phytases have been characterised from
different plant tissues but only the exceptional alkaline phytase
from lily pollen has phytate as the sole substrate (Scott and
Loewus, 1986; Baldi et al., 1988; Barrientos et al., 1994).
[0014] In 1997 the first plant phytase was cloned from Zea maize
(Maugenest et al., 1997). This enzyme was initially purified in
1993 (Laboure et al., 1993) and was described as a homo-dimer of a
38 kD polypeptide. An expression library was screened with
antibodies raised against the purified protein and this lead to the
identification of a full-length cDNA clone (phyS11). Two peptide
sequences determined from the purified maize enzyme were encoded by
the cDNA clone. The phyS11 clone was expressed in E. coli and a
polypeptide with the same migration, when using SDS-PAGE and
native-PAGE, as the maize phytase was obtained. However, no phytase
activity associated with the heterologous polypeptide could be
detected, even when the E. coli expressed protein was applied in 10
times higher concentrations than the detection level of the native
enzyme activity (Maugenest et al., 1997). Neither has genetic
transformation of maize with phyS11 constructs resulted in
expression of an active phytase enzyme (P. Perez Limagrain, pers.
Comm.).
[0015] The soybean phytase purified to apparent homogeneity from
10-day-old germinating cotelydons (Gibson and Ullah, 1988) is a
monomeric enzyme with a native molecular weight of 50 kD and it
migrates as two bands of 59 and 60 kD during SDS-PAGE (see table
1.). The enzyme activity is strongly inhibited by phosphate and 0.5
.mu.M phosphate renders the enzyme only 67% active (apparent
K.sub.i=18 .mu.M). This implies that the enzyme activity is tightly
regulated by product inhibition.
[0016] The soybean phytase is reportedly blocked N-terminally, but
an internal 18 amino acid from the enzyme was published in a 1990
review (Gibson and Ullah, 1990). This sequence (MHADQDYCANPQKYNXAI)
matches 100% with the sequence of soybean .beta.-amylase (result
not shown).
[0017] Another, N-terminal, soybean phytase sequence was published
in GB 2319030. The sequence disclosed in GB 2319030 is similar to
enzymes of the purple acid phosphatases (PAP) family, however this
is not described in the patent.
[0018] Phytase enzymes from wheat bran having an activity optimum
at pH 5.0 were purified and studied in detail by Nagai and
Funahashi in the early sixties (Nagai and Funahashi, 1962; Nagai
and Funahashi, 1963). Ten years later Lim and Tate (Lim and Tate,
1971, Lim and Tate, 1973) further published the presence and
partial purification of two wheat bran phytase enzymes that could
be separated on DEAE-cellulose but with identical molecular weights
of 47 kD. The two enzyme fractions F1 and F2 differed by their pH
optimum for activity of 5.6 and 7.2, respectively. The phytase
activity of fraction F1 with a pH optimum at 5.6 (Lim and Tate,
1973) had many similarities to the earlier described phytase
activity (Nagai and Funahashi, 1963) with an optimum at about 5.0,
but the F1 activity was inhibited by phosphate and the latter was
not inhibited at all. The products from the hydrolysis of phytate
by the F1 and F2 enzyme preparations were analysed (Lim and Tate,
1973) and it was found that although 6-phytase (the product being
D-ins 1,2,3,5,6) was the primary activity of both fractions, the F2
fraction also exhibited 5- and 2-phytase activity (Irving,
1980).
[0019] Nakano et al., 1999 purified and biochemically characterized
two wheat phytase enzymes from the "Nourin 61" wheat variety. The
N-terminal sequence of both enzymes were determined to be 13 amino
acid residues long having one unknown amino acid. A Swiss-Plot
database examination did not reveal homologue sequences. The
inventors did not disclose any gene or cDNA sequences.
[0020] In 1997 Nakano et al. purified and biochemically
characterized three N-terminal sequences from wheat bran
isoenzymes. Homologue sequences, gene or cDNA sequences were not
described.
[0021] Any PCR based cloning strategy requires both a forward and a
reverse primer. The N-terminal amino acid sequence can potentially
be used to construct a forward primer, but no specific reverse
primer can be made. It is therefore necessary to use a strategy
using one unspecific primer in combination with the specific
forward primer. Although such strategies exist (WALK-PCR, TAIL-PCR)
they do far from always prove successful and more importantly they
have as an absolute requirement that at least two nested
non-degenerate primers can be constructed from the known sequence,
which is not possible from the N-terminal amino acid sequence
published by Nakano et al. 1999. This is because of the high codon
degeneracy of the amino acids present in the amino terminal
polypeptide (N-terminal sequence of Nakano et al.). For example,
the amino acids arginine, serine and leucine are each represented
by six codons in the standard genetic code, while the amino acids
valine, threonine, proline, glycine, alanine are represented by
four codons each. Furthermore, the fourth residue from the
N-terminal is unknown Xaa, which altogether means these amino acids
constitute 12 of the 13 residues in the amino terminal polypeptide
sequence. Reverse transcription coupled PCR, RT-PCR on mRNA is
inefficient and by no means trivial for fragment sizes over 700
bases, thus demanding internal sequences for primer design.
[0022] Alternative strategies are based on hybridisation screening
of cDNA or genomic libraries, but a highly degenerate
oligonucleotide of a maximum of 39 bases is not sufficient to do
this
[0023] Although it has been the aim of several researchers to
obtain phytase sequence information from cereals, such as wheat and
barley, it is not until now that an isolated phytase from wheat is
presented, and specific wheat phytase sequences are disclosed along
with cloned wheat and barley phytase encoding nucleotide
sequences.
SUMMARY
[0024] Accordingly, it is an object of the present invention to
provide a phytase capable of being produced in large amounts.
[0025] In one aspect of the present invention a polypeptide having
affinity for the substrate phytate (1,2,3,4,5,6
myo-inositol-hexakisphosphate, phytic acid), comprising an amino
acid sequence as given in SEQ ID NO: 1 or an equivalent thereof,
having a sequence identity of SEQ ID NO: 1 of at least 70% and at
least one or more of the amino acid sequences as given in SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ
ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 13, or an equivalent thereof
having a sequence identity with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID
NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or
SEQ ID NO: 13 of at least 70% is provided.
[0026] Further, the invention relates to a DNA fragment (SEQ ID
NO.:11, and SEQ ID NO.:12) encoding said polypeptides and cDNA
fragments (SEQ ID NO.:9) encoding said polypeptides.
[0027] Additionally, an expression cassette comprising said DNA
fragments is described by the invention.
[0028] It is furthermore an object of the present invention to
provide for a cell which is capable of expressing a polypeptide and
which is transformed with an expression cassette as defined by the
invention.
[0029] Furthermore, it is an object of the invention to provide a
method of producing a polypeptide having affinity for the substrate
phytate (1,2,3,4,5,6 myo-inositol-hexakisphosphate, phytic acid)
comprising the steps of: [0030] short time extraction of the
polypeptide, [0031] obtaining an extract, [0032] subjecting the
extract to purification steps, [0033] purifying the polypeptide
from said extract, [0034] obtaining the polypeptide.
[0035] In an additional embodiment of the present invention a
method of producing a polypeptide having affinity for the substrate
phytate (1,2,3,4,5,6 myo-inositol-hexakisphosphate, phytic acid),
wherein a cell which contains a recombinant expression vector
comprising a DNA fragment encoding a polypeptide as defined above,
is cultured in a suitable medium under conditions which promote the
expression of the polypeptide, and where the polypeptide is
recovered from the culture is provided for.
[0036] The present invention also relates to a product obtained by
the methods as defined above and the invention describes the use of
the polypeptides as defined by the invention.
[0037] Another object of the present invention is disclosing a
transgenic plant or part thereof, wherein said plant or part
thereof have been genetically modified to comprise a polypeptide as
defined in by the invention.
DRAWINGS
[0038] FIG. 1: A) shows the SDS-PAGE separation of proteins in the
fractions from gelfiltration. Lane 2-9: 100 .mu.l of each fraction
13-20, lane 1: M12 molecular weight standards loaded with
approximately 0.5 .mu.g protein in each band. The phytase activity
of the fractions is given in the top of the lanes. B) is the
visualisation by silver staining gel after SDS-PAGE of the pool of
highly purified wheat bran phytase. N-terminal amino acid sequence
and tryptic mass fingerprints have been obtained for several of the
visible bands in lane 2. The two (broad) high MW and the two low MW
bands each has identical N-terminal sequences. The lower peptides
have been identified as .gamma.-conglutin homologues (Johansen and
Rasmussen, in prep). The upper two bands are the wheat bran
phytase. M12 molecular weight standards.
[0039] FIG. 2: shows a pH optimum curve for the highly purified
wheat bran phytase. Buffers used are marked at different
pH-intervals as indicated in the legend.
[0040] FIG. 3: shows a temperature optimum curve for wheat bran
phytase.
[0041] FIG. 4: the effect of various ions on the activity of the
highly purified wheat bran phytase. Activities measured in the
absence of additions to the reaction mix were set to 100%. The
values are averages of two measurements.
[0042] FIG. 5: depicts the isoelectric focusing of the purified
wheat bran phytase. A double band at pH 7.4 is visible after
incubation with 1-naphtylphosphate and Farst Garnet GBC.
[0043] FIG. 6: westernblot analysis of wheat bran proteins on PVDF
membrane. The membrane was cut in two after the transfer proteins:
lanes 1, 2, and 3 were coomassie blue stained, lanes 4, 5, and 6
were incubated with primary antibodies against the N-terminal of
the phytase polypeptide and alkaline phosphatase conjugated
secondary antibodies. Loading: lane 1, M12 MW markers (kD indicated
to the left); lanes 2, 250 ng and 4, 125 ng purified phytase; lanes
3 and 5, 2 .mu.l semi-purified preparation; lane 6, SeeBlue MW
markers. The arrow indicates the position of the phytase bands.
[0044] FIG. 7: shows the MALDI-TOF MS analysis of the tryptic
fragments of the wheat bran phytase. MALDI-TOF MS analysis of the
tryptic fragments of the wheat bran phytase. The peaks of 1046.54
and 2465.2 are internal standards. Peaks found in the fingerprint
of both the 56 kD and the 66 kD bands are indicated by an * next to
the mass.
[0045] FIG. 8: ClustaIW formatted alignment of the A) N-terminal or
internal B) and C) fragment of the translated sequence of three
putative PAP gene sequences from A. thaliana with a degree of
identity with the wheat bran phytase amino acid sequences. The
kidney bean sequence is included for comparison and the numbers of
residues for each of the fragments, when referring to the kidney
bean sequence are: A) Val.sub.16-Asp.sub.63, B)
Thr.sub.145-Leu.sub.163, and C) Val.sub.276-Tyr.sub.290. Accession
numbers are: kidney bean CAA04644, Arabidopsis1 ACC04486,
Arabidopsis2 CAB36834, Arabidopsis3 AC012395_20.
[0046] FIG. 9: shows the structure of phytate where the numbering
is according to the D-configuration.
DETAILED DESCRIPTION OF THE INVENTION
[0047] The present invention relates to a phytase polypeptide and
DNA fragments encoding a phytase polypeptide, in particular the
invention discloses the production and isolation of a wheat and/or
barley phytase. However, the invention is not limited to a phytase
isolated from wheat, but concerns any phytase having the
characteristics described by the invention. By the term phytase is
meant a polypeptide as described in the introductory part having
affinity for phytate. The phytase may have affinity for other
substrates as well, as long as the phytate affinity is of an
adequate size. The phytate affinity may be examined by using the
method described in the examples under purification of wheat bran
phytase. Thus a polypeptide or enzyme having affinity for phytate
is any polypeptide or enzyme having phytate affinity and being
capable of effecting the liberation of inorganic phospate or
phosphorous from various myo-inositol phosphates. Examples of such
myo-inositol phosphates (phytase substrates) are phytic acid and
any salt thereof, such as sodium phytate or potassium phytate or
mixed salts, or any stereoisomer of the mono-, di-, tri-, tetra- or
penta-phosphate of myo-inositol.
[0048] The definition of "a polypeptide or enzyme" also includes
fused polypeptides or cleavable fusion polypeptides in which
another polypeptide is fused at the N-terminus or the C-terminus of
the polypeptide or fragment thereof. A fused polypeptide is
produced by fusing a nucleic acid sequence (or a portion thereof)
encoding another polypeptide to a nucleic acid sequence (or a
portion thereof) of the present invention. Techniques for producing
fusion polypeptides are known in the art, and include ligating the
coding sequences encoding the polypeptides in frame and ensuring
that expression of the fused polypeptide is controlled by the same
promoter(s) and terminator. The polypeptide is preferably an
isolated polypeptide, which is meant to mean a polypeptide being
essentially free of other non-phytase polypeptides, such as at
least 20% pure, preferably at least 40% pure, more preferably at
least 60% pure, such as at least 80% pure, more preferably at least
90% pure, more preferably at least 95% pure as determined by
SDS-PAGE.
[0049] In the context of the present invention any amino acid(s)
designated: Xaa as mentioned in the amino acid sequence listings in
the present text are defined as being any amino acid.
[0050] In one embodiment of the present invention the polypeptide
is a phytase having affinity for phytate, comprising an amino acid
sequence as given in SEQ ID NO: 1 or an equivalent thereof having a
sequence identity with SEQ ID NO: 1 of at least 70%, such as 80%
and at least one or more of the amino acid sequences as given in
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 8,
SEQ ID NO: 10, or SEQ ID NO:12, or an equivalent thereof having a
sequence identity with SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ
ID NO: 6, SEQ ID NO: 8, SEQ ID NO:9, or SEQ ID NO:12 of at least
70%, such as at least 80%, for example at least 85%, such as at
least 90%, for example at least 95%.
[0051] The equivalent may be obtained by addition, substitution or
deletion of at least one amino acid.
[0052] A functional equivalent of a polypeptide of the invention is
to be understood as any part (or fragment) or any mimic having
affinity for phytate. A "functional equivalent" is defined as:
[0053] i) equivalents comprising an amino acid sequence capable of
being recognised by an antibody also capable of recognising the
predetermined amino acid sequence, and/or [0054] ii) equivalents
comprising an amino acid sequence capable of binding to a receptor
moiety also capable of binding the predetermined amino acid
sequence, and/or [0055] iii) equivalents having at least a
substantially similar or higher binding affinity to phytate as at
least a polypeptide of the invention comprising said predetermined
amino acid sequence.
[0056] According to the present invention a functional equivalent
of a polypeptide of the invention or fragments thereof may be
obtained by addition, substitution or deletion of at least one
amino acid in the polypeptide sequence.
[0057] Examples of equivalents comprising one or more conservative
amino acid substitutions including one or more conservative amino
acid substitutions within the same group of predetermined amino
acids, or a plurality of conservative amino acid substitutions,
wherein each conservative substitution is generated by substitution
within a different group of predetermined amino acids.
[0058] In another embodiment of the invention the polypeptide
comprises an N-terminal amino acid sequence as described in SEQ ID
NO:1, or an equivalent thereof having a sequence identity with SEQ
ID NO:1 of at least 70% such as 80% and at least one or more of the
amino acid sequences as given in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID
NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or
SEQ ID NO: 13, or an equivalent thereof having a sequence identity
with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID
NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 13 of at least
70%.
[0059] The equivalent may be obtained by addition, substitution or
deletion of at least one amino acid.
[0060] A further embodiment relates to a polypeptide having an
amino acid sequence as given in SEQ ID NO:1 or an equivalent
thereof having a sequence identity with SEQ ID NO:1 of at least
70%, such as at least 80%, for example at least 85%, such as at
least 90%, for example at least 95%. and an amino acid sequence as
given in SEQ ID NO: 2 or an equivalent thereof having a sequence
identity with SEQ ID NO:2 of at least 70%, such as at least 80%,
for example at least 85%, such as at least 90%, for example at
least 95%.
[0061] The equivalent may be obtained by addition, substitution or
deletion of at least one amino acid.
[0062] In yet a further embodiment a polypeptide having an amino
acid sequence of given in SEQ ID NO:1 or an equivalent thereof
having a sequence identity with SEQ ID NO:1 of at least 70%, such
as at least 80%, for example at least 85%, such as at least 90%,
for example at least 95%. and an amino acid sequence as given in
SEQ ID NO: 3 or an equivalent thereof having a sequence identity
with SEQ ID NO:3 of at least 70%, such as at least 80%, for example
at least 85%, such as at least 90%, for example at least 95%.
[0063] In another embodiment given in SEQ ID NO:1 or an equivalent
thereof having a sequence identity with SEQ ID NO:1 of at least
70%, such as at least 80%, for example at least 85%, such as at
least 90%, for example at least 95%. and an amino acid sequence as
given in SEQ ID NO:4 or an equivalent thereof having a sequence
identity with SEQ ID NO:4 of at least 70%, such as at least 80%,
for example at least 85%, such as at least 90%, for example at
least 95%.
[0064] The invention also relates to a polypeptide having an
internal amino acid sequence in given in SEQ ID NO:1 or an
equivalent thereof having a sequence identity with SEQ ID NO:1 of
at least 70%, such as at least 80%, for example at least 85%, such
as at least 90%, for example at least 95%. and an amino acid
sequence as given in SEQ ID NO: 6 or an equivalent thereof having a
sequence identity with SEQ ID NO:5 of at least 70%, such as at
least 80%, for example at least 85%, such as at least 90%, for
example at least 95%.
[0065] In a further embodiment the invention discloses a
polypeptide having an amino acid sequence as given in SEQ ID NO:1
or an equivalent thereof having a sequence identity with SEQ ID
NO:1 of at least 70%, such as at least 80%, for example at least
85%, such as at least 90%, for example at least 95%. and an amino
acid sequence as given in SEQ ID NO: 8 or an equivalent thereof
having a sequence identity with SEQ ID NO:7 of at least 70%, such
as at least 80%, for example at least 85%, such as at least 90%,
for example at least 95%.
[0066] In yet another embodiment the invention discloses a
polypeptide having an amino acid sequence as given in SEQ ID NO:1
or an equivalent thereof having a sequence identity with SEQ ID
NO:1 of at least 70%, such as at least 80%, for example at least
85%, such as at least 90%, for example at least 95%. and an amino
acid sequence as given in SEQ ID NO: 10 or an equivalent thereof
having a sequence identity with SEQ ID NO:9 of at least 70%, such
as at least 80%, for example at least 85%, such as at least 90%,
for example at least 95%.
[0067] In a further embodiment the invention discloses a
polypeptide having an amino acid sequence as given in SEQ ID NO:1
or an equivalent thereof having a sequence identity with SEQ ID
NO:1 of at least 70%, such as at least 80%, for example at least
85%, such as at least 90%, for example at least 95%. and an amino
acid sequence as given in SEQ ID NO: 13 or an equivalent thereof
having a sequence identity with SEQ ID NO:12 of at least 70%, such
as at least 80%, for example at least 85%, such as at least 90%,
for example at least 95%.
[0068] In a preferred embodiment of the invention the polypeptide
is having an amino acid sequence of a wheat phytase comprising
sequences as given in SEQ ID NO:1 or an equivalent thereof having a
sequence identity with SEQ ID NO:1 of at least 70%, such as at
least 80%, for example at least 85%, such as at least 90%, for
example at least 95%. and an amino acid sequence as given in SEQ ID
NO: 2 or an equivalent thereof having a sequence identity with SEQ
ID NO:2 of at least 70%, such as at least 80%, for example at least
85%, such as at least 90%, for example at least 95%. and a
polypeptide having an amino acid sequence as given in SEQ ID NO:3
or an equivalent thereof having a sequence identity with SEQ ID
NO:3 of at least 70%, such as at least 80%, for example at least
85%, such as at least 90%, for example at least 95%.
[0069] In a further preferred embodiment the polypeptide is having
an amino acid sequence of a wheat phytase comprising sequences as
given in SEQ ID NO:1 or an equivalent thereof having a sequence
identity with SEQ ID NO:1 of at least 70%, such as at least 80%,
for example at least 85%, such as at least 90%, for example at
least 95%. and an amino acid sequence as given in SEQ ID NO: 5 or
an equivalent thereof having a sequence identity with SEQ ID NO:4
of at least 70%, such as at least 80%, for example at least 85%,
such as at least 9p%, for example at least 95%. and a polypeptide
having an amino acid sequence as given in SEQ ID NO:3 or an
equivalent thereof having a sequence identity with SEQ ID NO:3 of
at least 70%, such as at least 80%, for example at least 85%, such
as at least 90%, for example at least 95%.
[0070] In yet a further preferred embodiment the polypeptide is
having an amino acid sequence of a wheat phytase comprising
sequences as given in SEQ ID NO:1 or an equivalent thereof having a
sequence identity with SEQ ID NO:1 of at least 70%, such as at
least 80%, for example at least 85%, such as at least 90%, for
example at least 95%. and an amino acid sequence as given in SEQ ID
NO: 6 or an equivalent thereof having a sequence identity with SEQ
ID NO:5 of at least 70%, such as at least 80%, for example at least
85%, such as at least 90%, for example at least 95%. and a
polypeptide having an amino acid sequence as given in SEQ ID NO:3
or an equivalent thereof having a sequence identity with SEQ ID
NO:3 of at least 70%, such as at least 80%, for example at least
85%, such as at least 90%, for example at least 95%.
[0071] In yet another embodiment the invention discloses a DNA
molecule having a nucleotide sequence as given in SEQ ID NO:8 or an
equivalent thereof having a sequence identity with SEQ ID NO:8 of
at least 70%, such as at least 80%, for example at least 85%, such
as at least 90%, for example at least 95%. is provided for. The
equivalent may be obtained by addition, substitution or deletion of
at least one nucleotide.
[0072] The invention also relates to a polypeptide having an amino
acid sequence as given in SEQ ID NO:9 or an equivalent thereof
having a sequence identity with SEQ ID NO:9 of at least 80%, such
as 90% is provided for. The equivalent may be obtained by addition,
substitution or deletion of at least one amino acid.
[0073] In a further aspect a polypeptide having an amino acid
sequence as given in SEQ ID NO:12 or an equivalent thereof having a
sequence identity with SEQ ID NO:12 of at least 80%, such as 90% is
provided for. The equivalent may be obtained by addition,
substitution or deletion of at least one amino acid.
[0074] In a further embodiment the invention discloses a DNA
molecule having a nucleotide sequence as given in SEQ ID NO: 11 or
an equivalent thereof having a sequence identity with SEQ ID NO: 11
of at least 80%, such as 90% is provided for. The equivalent may be
obtained by addition, substitution or deletion of at least one
nucleotide.
[0075] Additionally, the invention discloses a DNA molecule having
a nucleotide sequence as given in SEQ ID NO: 12 or an equivalent
thereof having a sequence identity with SEQ ID NO: 12 of at least
80%, such as 90% is provided for. The equivalent may be obtained by
addition, substitution or deletion of at least one nucleotide.
[0076] The DNA molecules of the invention encoding a phytase
polypeptide as given in SEQ ID NO: 9, 11 and 12 may be modified to
optimise the codon usage for improved expression in a particular
organism, such as bacteria, fungi, and plants.
[0077] When the amino acid sequences of the invention comprise a
substitution of one amino acid for another, such a substitution may
be a conservative amino acid substitution as defined herein above.
Sequences according to the present invention may comprise more than
one such substitution, such as e.g. two conservative amino acid
substitutions, for example three or four conservative amino acid
substitutions, such as five or six conservative amino acid
substitutions, for example seven conservative amino acid
substitutions. Substitutions can be made within any one or more
groups of predetermined amino acids. Conservative substitutions may
be introduced in any position of a preferred predetermined
polypeptide or fragment thereof. It may however also be desirable
to introduce non-conservative substitutions, particularly, but not
limited to, a non-conservative substitution in any one or more
positions.
[0078] The addition or deletion of an amino acid may be an addition
or deletion of from 2 to preferably 7 amino acids, such as from 2
to 5 amino acids, for example from 2 to 3 amino acids. However,
additions of more than 7 amino acids, such as additions from 8 to
10 amino acids, are also comprised within the present
invention.
[0079] In the context of the invention the term a functional
equivalent relates to a sequence or a polypeptide comprising a
sequence possessing a corresponding property as the polypeptides
comprising the sequences mentioned in the present invention, but
wherein one or more amino acids have been substituted with others.
Preferably a functional equivalent contains substitutions, i.e.
where one or more amino acids are substituted by an amino acid
having similar properties.
[0080] The amino acids suitable for substitutions may include those
having functionally similar side chains. For example, hydrophobic
residues: e.g. glycine, alanine, valine, leucine, isoleucine and
methionine may replace another such residue. Similarly,
conservative substitutions may involve interchanging hydrophilic
residues: (e.g.:
[0081] arginine and lysine, glutamine and aspargine, threonine and
serine), basic reduces (e.g., lysine, arginine and histidine),
and/or acidic residues (e.g., aspartic acid and glutamic acid).
Functional equivalents may also, or alternatively, be modified by
for example the deletion or addition of amino acids, or the
chemical modification of amino acids, as long as the function of
the polypeptide is preserved.
[0082] The isolated wheat bran polypeptide comprising one or more
sequences of the present invention, including any variants and
functional equivalents thereof, may in one embodiment comprise less
than 250 amino acid residues, such as less than 225 amino acid
residues, for example less than 200 amino acid residues, such as
less than 175 amino acid residues, for example less than 150 amino
acid residues, such as less than 125 amino acid residues, for
example less than 100 amino acid residues.
[0083] In a more preferred embodiment the polypeptide comprises at
least two of the sequences described. Preferably the polypeptide
comprises SEQ ID NO:1 or an equivalent thereof and at least one of
the other sequences or equivalents thereof.
[0084] The equivalents are at least 70% identical with the
sequences shown herein, such as at least 75% identical, preferably
at least 80% identical, such as at least 85% identical, such as at
least 90% identical, for example at least 95% identical.
[0085] Where a particular polypeptide is said to have a specific
percent identity to a reference polypeptide of a defined length.
Thus, a polypeptide that is 50% identical to a reference
polypeptide that is 20 amino acids long can be a 10 amino acid
polypeptide that is completely identical to a 10 amino acid long
portion of the reference polypeptide. It might also be a 20 amino
acid long polypeptide which is 50% identical to the reference
polypeptide over its entire length. The degree of identity between
the sequences of the invention and their equivalents may be
determined by commercially available computer software, such as
MacVector.
[0086] In yet a further embodiment of the invention the polypeptide
is capable of binding a monoclonal anti-body raised against a
polypeptide having the sequence of SEQ ID NO: 1 and at least one or
more of the amino acid sequences as given in SEQ ID NO: 2, SEQ ID
NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or
SEQ ID NO: 13.
[0087] According to the invention monoclonal antibodies may also be
raised against a polypeptide comprising the SEQ ID NO:1 and at
least one or more of the amino acid sequences as given in SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ
ID NO: 10, or SEQ ID NO: 13.
[0088] In a preferred embodiment the polypeptide comprise one or
more of the sequences listed above and bind the antibody as defined
above.
[0089] The polypeptide of the invention has various biochemical
characteristics. Accordingly, the denatured polypeptide of the
invention preferably has a molecular weight of 50-75 kDa,
preferably 53-70 kDa, more preferable 55-68 kDa as determined by
SDS-PAGE (Sodium dodecyl sulphate PolyAcrylamide Gel
Electrophoresis).
[0090] Furthermore, the polypeptide of the invention has a native
molecular weight of 35-70 kDa as determined by gelfiltration, such
as 40-66, for example 45-60.
[0091] The polypeptide preferably has a substrate affinity of
0.05-0.50 mM, such as 0.08-0.45, for example 0.10-0.35, such as
0.14-0.25.
[0092] The polypeptide preferably has a substrate affinity of
0.002-0.50 mM, such as 0.08-0.45, for example 010-0.35, such as
0.14-0.25. In one aspect of the invention the polypeptide has a
V.sub.max of 600-1000 U/mg, such as 700-900 U/mg, for example
750-850 U/mg. V.sub.max is defined as the maximum reaction
velocity, i.e. the maximal velocity obtained when all of the
phytase is in the form of the phytase-phytate complex. The
substrate affinity and the reaction velocity may be determined by
designing classical biochemical experiments, and measure the
concentration of the liberated phosphate as described below in the
experimentals section. The enzyme activity may be determined by
measuring the amount of released phosphate through the colour
intensity of a phosphate complex such as the yellow colour of the
phosphate-molybdate-vanadate complex. Alternatively the amount of
lower myo-inositol phosphates generated by the enzyme activity may
be quantified by HPLC-based methods.
[0093] In another aspect of the invention the polypeptide has an
isoelectric point of between 5.0-8.0, such as between 5.5-7.0.
[0094] The polypeptide according to the invention preferably has a
pH optimum of between 4 and 8, preferably between 5-7. This refers
to the polypeptide having an optimal activity in the above ranges
of pH values.
[0095] Also, the polypeptide of the invention, has an optimal
activity at a temperature between 30-65.degree. C., preferably
between 35-60.degree. C., and more preferably between 40-55.degree.
C. In the present context the term "optimal activity" is defined as
the optimal rate of which the enzyme/polypeptide of the invention
converts it substrate myo-inositol-1,2,3,4,5,6-hexakisphosphate or
phytic acid into inorganic phosphate and lower
myo-inositol-phosphates, such as
D-myo-inositol-1,2,3,5,6-hexakisphosphate,
D-myo-inositol-1,2,5,6-hexakisphosphate among others. In one
embodiment of the invention the polypeptide exhibits 6-phytase
activity (EC 3.1.3.26).
[0096] In a preferred embodiment the polypeptide has a
thermostability that is sufficient to withstand the heat processing
of fodder. This can be achieved by random muta-genesis and
selection in thermostabile microorganisms, or by point mutation of
selected amino acids to achieve better thermostabillity of the
polypeptide with the purpose of maintaining sufficient residual
activity for down stream use.
[0097] Another important purpose of the invention is to provide a
DNA fragment encoding a polypeptide according to the invention. The
invention also relates to a cDNA fragment encoding the polypeptide
of the invention.
[0098] The composition of the polypeptide sequences of the
invention is such that isolation of a DNA molecule encoding a
phytase polypeptide according to the invention has proved difficult
even to someone skilled in the art. This is because of the high
codon degeneracy of the amino acids present in the amino terminal
polypeptide (SEQ ID NO.:1) of the invention. For example, the amino
acids arginine and leucine are both represented by six codons in
the standard genetic code, while the amino acids valine, threonine,
proline, glycine, alanine are represented by four codons each.
Together these amino acids constitute 12 of the 15 residues in the
amino terminal polypeptide sequence.
[0099] In a preferred embodiment of the invention a DNA molecule
encoding a polypeptide according to the invention is therefore
isolated using a degenerate oligonucleotide primer derived from
reverse translation of the internal polypeptide of the invention
(SEQ ID NO.:2) and an oligonucleotide primer annealing to DNA
sequences of a barley EST clone (acc. No. BE602374). This clone can
be identified as putatively encoding a purple acid phosphatase by
its degree of identity to the carboxy-terminal part of the
Arabidopsis thaliana putative purple acid phosphatase (acc. No.
AAF20233), which itself shows partial conservation of the
polypeptide sequences of the invention (see FIG. 8). Employing the
polymerase chain reaction (PCR) using these oligonucleotide primers
and genomic DNA isolated from wheat as template will result in
isolation of a DNA molecule encoding the carboxy terminal part of
the polypeptide of the invention. DNA fragments encoding the
amino-terminal part of the polypeptide of the invention is isolated
by sequential PCR on restriction digested genomic wheat DNA to
which an adapter has been ligated using nested primers annealing to
the adapter and the carboxy-terminal part of the polypeptide of the
invention.
[0100] In one embodiment of the invention the complete genomic DNA
fragment or parts of it is preferably isolated using PCR with
oligonucleotide primers from the amino- and carboxy-terminal parts
on genomic DNA from wheat or barley.
[0101] In a further embodiment the complete cDNA fragment of the
invention or parts of it is preferably isolated using RT-PCR with
oligonuceotide primers from the amino-and carboxy-terminal parts on
RNA from immature wheat or barley kernels.
[0102] Furthermore, the DNA fragment of the invention may be
subjected to cloning procedures. This may involve excision and
isolation of a desired nucleic acid fragment comprising the nucleic
acid sequence encoding the polypeptide, insertion of the fragment
into a vector molecule, and incorporation of the recombinant vector
into a host cell where multiple copies or clones of the nucleic
acid sequence will be replicated.
[0103] The nucleic acid sequence may be of genomic, cDNA, RNA,
semisynthetic, synthetic origin, or any combinations thereof.
[0104] The invention further relates to a DNA construct comprising
the polypeptide as defined by the invention and an expression
cassette comprising the DNA construct. The expression vector
carrying the DNA construct of the invention may be any vector which
may conveniently be subjected to recombinant DNA procedures, and
the choice of vector will often depend on the host cell into which
it is to be introduced. Thus, the vector may be an autonomously
replicating vector, i.e. a vector which exists as an extra
chromosomal entity, the replication of which is independent of
chromosomal replication, for example a plasmid, a bacteriophage or
an extra chromosomal element, mini chromosome or an artifical
chromosome. Alternatively, the vector may be one which, when
introduced into a host cell, is integrated into the host cell
genome and replicated together with the chromosome(s) into which it
has been integrated. Thus, the present invention further relates to
a cell which is capable of expressing a polypeptide and which is
transformed with an expression cassette as defined by the
invention.
[0105] The cell of the invention either comprising a DNA construct
or an expression vector according to the invention as defined above
is advantageously used as a host cell in the recombinant production
of a protein variant according to the invention. The cell may be
transformed with the DNA construct, for example by integrating the
DNA construct in the host chromosome. Integration is considered to
be an advantage as the DNA fragment is more likely to be stably
maintained in the cell. Alternatively, the cell may be transformed
as described above for the expression vector.
[0106] According to the invention the host cell may be chosen from
mammal, avian, insect or plant cells, or it may be selected from
bacteria or fungi. The host cell is responsible for synthesising
and expressing the polypeptide according to the invention and an
expression cassette may transform the cell.
[0107] Preferably the host cell is a bacteria selected from gram
positive bacteria, such as Bacillus subtilis, Bacillus
licheniformis, Bacillus lentus, Bacillus brevis, Bacillus
stearothermophilus, Bacillus alkalophilus, Bacillus
amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus
lautus, Bacillus megaterium, Bacillus thuringiensis, or
Streptomyces lividans or Streptomyces murinus, or a gram negative
bacteria, such as E.coli.
[0108] More preferably the host cell of the present invention is of
fungal origin and is selected among the species of Saccharomyces or
Schizosaccharomyces, e.g. Saccharomyces cerevisiae. In a preferred
embodiment of the present invention the fungus may advantagerously
belong to the species of Aspergillus, e.g. Aspergillus oryzae or
Aspergillus niger or Pichia, e.g. Pichia pastoris. In a less
preferred embodiment the species of Fusarium, e.g. F. oxysporum may
be used as a host cell.
[0109] Most preferably the host cell is of plant origin, and may be
selected from any plant, such as a plant cell from wheat, barley,
rye, rye grass, spelt, oat, rice or maize.
[0110] Alternatively, the DNA fragment of the DNA construct may be
prepared synthetically by established standard methods.
[0111] It is a purpose of the present invention to provide for a
method of producing a recombinant polypeptide having affinity for
the substrate phytate (1,2,3,4,5,6 myo-inositol-hexakisphosphate,
phytic acid), wherein a cell which contains a recombinant
expression vector comprising a DNA fragment encoding a polypeptide
as defined by the invention, is cultured in a suitable medium under
conditions which promote the expression of the polypeptide, and
where the polypeptide is recovered from the culture.
[0112] According to the invention the polypeptide is being
synthesised and expressed in host cells. This is achieved by
culturing host cells capable of expressing a polypeptide in a
suitable culture medium to obtain expression, and possibly
secretion of the polypeptide. The polypeptide may be recovered from
the culture medium or from the cells or from the cell culture as a
whole.
[0113] Furthermore, the present invention describes a method of
producing a wheat bran or barley polypeptide having affinity for
the substrate phytate (1,2,3,4,5,6 myo-inositol-hexakisphosphate,
phytic acid) comprising the steps of. [0114] short time extraction
of the polypeptide, [0115] obtaining an extract, [0116] subjecting
the extract to purification steps, [0117] purifying the pblypeptide
from said extract, [0118] obtaining the polypeptide.
[0119] The plant material may be any plant material. In one
embodiment of the invention the plant material selected are wheat
roots. In a preferred embodiment of the invention the plant
material selected are wheat seeds.
[0120] In another embodiment the plant material may be selected
from barley roots. However, in a preferred embodiment the plant
material selected may be from barley seeds.
[0121] Once the polypeptide of the invention has been extracted
from the plant material chosen it is necessary to purify the
polypeptide from the extract. According to the invention the
purification of the polypeptide of the invention is performed by a
combination of different biochemical separation methods. In a
preferred embodiment of the invention the purification procedure is
performed stepwise as follows: ammonium sulphate precipitation,
filtration, dialysis, ion exchange chromatography, ultrogel,
resource S ion exchange, and Superose 12 gelfiltration. These are
all well established techniques in the art of protein purification
and leads to the stepwise separation of protein species in the
extract based on hydrophobicity, charge, affinity towards
hydroxyapatite and molecular size. The order of the individual
purification steps of the invention may be any order of the above
mentioned purification methods.
[0122] An essential feature of the purification process of the
present invention is the short time span in which the purification
is performed. Thus, in a preferred embodiment of the invention the
extraction protocol is characterised by a short extraction time. It
is desirable to have the extraction period being as short as
possible to avoid proteolysis. In the context of the present
invention a short time extraction may be a period of 60 minutes,
such as 45 minutes, for example 30 minutes. Another important
characteristic feature of the purification protocol of the present
invention is the inclusion of only aqueous solvents (the detailed
purification procedure is described below in the experimentals
section).
[0123] Having obtained the polypeptide according to the invention a
continuation of the method of producing a polypeptide defined by
the invention and described above comprises the steps of: [0124]
exposing the polypeptide to epitope specific antibodies raised
against said polypeptide, [0125] assessing the specificity by
Western blotting.
[0126] The epitope specific antibodies raised against the
polypeptide may be raised by the means of conventional procedures.
According to the invention a synthetic peptide corresponding to the
amino acid sequence of the wheat polypeptide of the invention were
conjugated to a carrier enzyme, such as the Keyhole limpet
hemocyanin protein, and polyclonal antibodies against the
polypeptide may be raised in for example a rabbit. By the term a
"carrier protein" is a scaffold structure, e.g. a polypeptide or a
polysaccharide, to which an immunogenic determinant is capable of
being associated. When the synthetic polypeptide of the invention
is obtained a carrier may be associated with the polypeptide. The
carrier may be either non-conjugated or conjugated. When the
carrier is conjugated, the polypeptide may be conjugated to said
carrier, or the carrier may be conjugated to said polypeptide. The
polypeptide may--in addition to a carrier--further comprise an
adjuvant for increasing the efficacy of the composition. Any
suitable adjuvant may be used in combination with the
polypeptide/carrier composition. In the present context the term
conjugated refers to an association formed between the polypeptide
and a carrier. The association may be a physical association
generated e.g. by the formation of a chemical bond, such as e.g. a
covalent bond, formed between the polypeptide and the carrier.
[0127] When using the methods described by the invention products
are obtained. The products of the invention relate to wheat bran
polypeptides having affinity for phytate.
[0128] A very important aspect of the invention is the use of a
polypeptide as defined in the invention.
[0129] In one aspect the polypeptide is used as an additive in
animal feeds. The polypeptide of the invention may be applied to
the feed, such as fodder-pellets. It is important that the phytase
polypeptide added to the fodder has the desirable properties of a
broad substrate specificity, a high specific activity and
resistance towards proteolysis. Since the production of
fodder-pellets is conducted under high temperatures it is an object
of the invention to provide a thermostable phytase polypeptide. By
adding phytase enzymes to animal feeds a reduction in the addition
of phosphorous may be obtained, leading to more environmentally
friendly animal feeds.
[0130] Applying the polypeptide of the invention as an additive in
food for human consumption is also a concern. Phytate degradation
is not merely a concern in relation to farm animals but also in
relation to human nutrition.
[0131] A number of reports on anticancer effects of phytase have
been published during the last few years. In an experiment on rat,
it has been shown that when labelled phytase is given in the
drinking water is rapidly taken up and distributed in the body.
Here it leads to a 33.5% reduction in mammary tumor incidences as
compared to a control group (Shamsuddin and Vucenik, 1999). The
myo-inositol moiety itself have a similar effect on lung and liver
carcinogenesis in mice and it is therefore suspected that a degree
of phosphorylation or dephosphorylation of the inositol compounds
must occur in the cells (Nishino et al., 1999). These reports are
emphasising the importance of an effective degradation of
phytate-aggregates in the food in order to improve the uptake of
healthy and necessary food-components.
[0132] Application of fungal phytase enzymes in the preparation of
foods for humans have been reported. The present invention thus
stresses the use of wheat bran phytase according to the invention
as an industrial processing enzyme.
[0133] In one embodiment of the invention the polypeptide of the
invention is used to extract proteins from rice bran. The
processing of rice bran to generate a protein isolate that may be
used for e.g. the formulation of infant food is within the scope of
the invention. The processing rate may increase significantly when
incubation with the polypeptide of the invention is included in the
procedure. Clearly, the present invention presents advantages in
industrial applications, such as turning inexpensive wheat bran
into high profit products.
[0134] In a further aspect the present invention relates to the
generation of high-phytase rice. This aspect not only may improve
phosphor and mineral but also protein digestibility. This is a
crucial fact when attempting to raise the nutritional value of
human diets in countries where rice is a major part of the diet. In
such countries particularly zinc and iron deficiencies are major
problems that the present invention may help alleviate.
[0135] Phosphorus is a limiting factor for plant growth in many
parts of the world. Phosphorus is found in the soil both as an
inorganic and organic form in the soils. The major part of the
organic phosphate is inositol phosphates which is poorly or not
utilized by many plant species. This is because the interaction of
phytate with soil-matters renders inositol phosphates inaccessible
for plant up-take, or the plant root does not provide mechanisms
for the release and subsequent uptake of inositol phosphates. It is
within the scope of the present invention to overcome this problem
by extracellular secretion of wheat or barley phytase from roots of
plants which have been transformed with a phytase gene of the
invention. This will enable these plants to utilize phosphorus from
inositol phosphates and or phytate and thereby sustain the plant
growth organic phosphorus pools in the soil. The phytase DNA may be
under control of a root specific promotor.
[0136] The wheat and barley phytase encoding nucleotide sequences
according to the invention may allow for transformation of plants.
The level of intrinsic phytase activity in many plants is
relatively low compared to the phytate content, and not sufficient
for adequate digestibility of phosphorus in monogastric animals.
Thus, according to the invention the use of the polypeptide may be
in a transgenic plant or part thereof. In a further aspect the
invention relates to the use of a transgenic plant or part thereof,
wherein said plant or part thereof have been genetically modified
to comprise a polypeptide as defined in the invention. The
expression of the wheat bran phytase and barley phytase could under
the control of a seed specific promoter be synthesised and stored
in large quantities in the mature grain. Wheat bran and barley
phytases have the advantage of their natural design for storage in
the seeds.
[0137] It is envisioned that the use of the polypeptide of the
invention may be in any transgene plant for which it is desirable
to obtain additional phytase activity.
[0138] The transgenic plant may be dicotyledonous or
monocotyledonous, for short a dicot or a monocot. Of primary
interest are such plants which are potential food or feed
components and which comprise phytic acid. A normal phytic acid
level of feed components is 0.1-30 g/kg, or more usually 0.5-25
g/kg, most usually 0.5-18 g/kg. Examples of monocot plants are
grasses, such as meadow grass (blue grass, Poa), forage grass such
as festuca, lolium, temperate grass, such as Agrostis, and cereals,
e.g. wheat, oats, rye, barley, rice, sorghum and maize (corn).
[0139] Examples of dicot plants are legumes, such as lupins, pea,
bean and soybean, and cruciferous (family Brassicaceae), such as
cauliflower, oil seed rape (canola) and the closely related model
organism Arabidopsis thaliana.
[0140] Preferably, the plant or plant part, e.g. the seeds, are
ground or milled, and possibly also soaked before being added to
the food or feed or before the use, e.g. intake, thereof, with a
view to adapting the speed of the enzymatic degradation to the
actual use. If desired, the enzyme produced by the plant may also
be recovered from the plant. In certain cases the recovery from the
plant is to be preferred with a view to securing a heat stable
formulation in a potential subsequent pelleting process.
[0141] Examples of plant parts are stem, callus, leaves, root,
fruits, seeds, tubers etc. But also any plant tissue is included in
this definition.
[0142] Any plant cell, whatever the tissue origin, is included in
the definition of plant cells above. Also included within the scope
of the invention are the progeny of such plants, plant parts and
plant cells. The skilled person will known how to construct a DNA
expression construct for insertion into the plant in question,
paying regard i.e. to whether the transcript should be expressed in
a tissue specific way. Of relevance for this evaluation is the
stability (pH-stability, degradability by endogenous proteases
etc.) of the phytase in the expression compartments of the plant.
The skilled artisan will also be able to select appropriate
regulatory sequences such as promoter and terminator sequences, and
signal or transit sequences if required (Tague et al, 1988).
[0143] The plant, plant part etc. can be transformed with the DNA
construct using any known method. An example of such method is the
transformation by a viral or bacterial vector such as bacterial
species of the genus Agrobacterium genetically engineered to
comprise the gene encoding the phytase of the invention. Also
methods of directly introducing the phytase DNA into the plant cell
or plant tissue are known in the art, e.g. micro injection and
electroporation (Gasser et al, 1993; Potyrkus, 1990; Shimamoto et
al, 1989), or particle bombardment (microscopic gold or tungsten
particles coated with the transforming DNA) of embryonic calli or
developing embryos (Christou, 1992; Shimamoto, 1994
[0144] Following the transformation, the transformants are screened
by using any method known to the skilled person, following which
they are regenerated into whole plants.
[0145] These plants etc. as well as their progeny then carry the
phytase encoding DNA as a part of their genetic equipment.
Agrobacterium tumefaciens mediated gene transfer is the method of
choice for generating transgenic dicots (for review Hooykas &
Schilperoort, 1992). The method of choice for generating transgenic
monocots is particle bombardment.
[0146] Also, other systems for the delivery of free DNA into
plants, include viral vectors (Joshi & Joshi, 1991), protoplast
transformation via polyethylene glycol or electroporation (for
review see Potyrkus, 1991), or microinjection of DNA into mesophyll
protoplasts (Crossway et al., 1986).
[0147] In general, the cDNA or gene encoding the phytase of the
invention is placed in an expression cassette (e.g. Pietrzak et
al., 1986) consisting of a suitable promoter active in the target
plant and a suitable terminator (termination of transcription).
This cassette will be transformed into the plant as such in case of
monocots via particle bombardment. In the case of dicots the
expression cassette is placed first into a suitable vector
providing the T-DNA borders and a suitable selection marker which
in turn are transformed into Agrobacterium tumefaciens. Dicots will
be transformed via the Agrobacterium harbouring the expression
cassette and selection marker flanked by T-DNA following standard
protocols (e.g. Akama et al., 1992). The transfer of T-DNA from
Agrobacterium to the Plant cell has been recently reviewed (Zupan
& Zambryski, 1995). Vectors for plant transformation via
Agrobacterium are commercially available or can be obtained from
many labs that construct such vectors (e.g. Deblaere et al., 1985;
for review see Klee et al., 1987).
[0148] Available plant promoters: depending on the process under
manipulation, organ- and/or cell-specific expression as well as
appropriate developmental and environmental control may be
required. For instance, it is desirable to express a phytase cDNA
in maize endosperm etc. The most commonly used promoter has been
the constitutive 35S-CaMV promoter Franck et al., 1980). Expression
will be more or less equal throughout the whole plant. This
promoter has been used successfully to engineer herbicide- and
pathogen-resistant plants (for review see Stitt & Sonnewald,
1995). Organ-specific promoters have been reported for storage sink
tissues such as seeds, potato tubers, and fruits (Edwards &
Coruzzi, 1990), and for metabolic sink tissues such as meristems
(Ito et al., 1994).
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[0216] The following is an example of the purification and
production of a wheat bran phytase according to the invention.
Materials
[0217] All reagents used were laboratory grade, all water used
filtered through Millipore system. Havnemollen (.ANG.rhus) provided
commercially available wheat bran.
Purification of Wheat Bran Phytase
[0218] Extraction of phytase activity from 2 kg wheat bran was
performed using 10 l H.sub.2O, 200 g PVPP and 5 mM
.beta.-mercaptoethanol. The suspension was stirred for 30 min. at
room temperature and then filtrated through a fine mesh. The
filtrate was adjusted to 50 mM NaAc pH 4.5 (buffer A) and
centrifuged for 20 min at 8.000 rpm in a GSA rotor at 4.degree. C.
in a Sorwall centrifuge, to precipitate starch and insoluble
protein. The supernatant was adjusted to 30% ammonium sulphate and
incubated for 2 hours prior to centrifugation for 20 min at 8.000
rpm in a GSA rotor at 4.degree. C. The resulting supernatant was
adjusted to 60% ammonium sulphate, incubated and spun again. The
pellet was solubilised in 200 ml buffer A and dialysed twice
towards buffer A. The dialysed proteins were then diluted to 1 l
with buffer A and loaded on a 600 ml SP sepharose (Pharmacia)
column that was equilibrated in the same buffer. Bound proteins
were eluted with 6 l of a linear gradient of 50 mM NaAc, 1 M NaCl,
pH 4.5 (buffer B). The resulting fractions were assayed for phytase
activity using the assay of Engelen et al (Engelen et al., 1994)
(adapted for eppendorf tubes). Fractions containing phytase
activity were pooled, the protein precipitated by the addition of
ammonium sulphate, the pellet resuspended and the buffer exchanged
using HiTrap desalting columns (Pharmacia) to an unbuffered 1 mM
NaCl solution. The resulting 50 ml extract was loaded on a 100 ml
Ultrogel (hydroxy apatite, Sigma) column and the phytase activity
was collected in the flow-through and initial wash. The phytase
containing fraction was immediately adjusted to 50 mM NaAc pH 4.5
and 10% glycerol. The phytase-pool was then fractionated on a 6 ml
Resource S column (Pharmacia) by elution with a linear gradient of
buffer B. The procedure was repeated on a 1 ml Resource S column
(Pharmacia). The phytase containing fractions were pooled and
concentrated on Centricon 10 (Amicon) filters, loaded in 200 .mu.l
portions on a Superose gelfiltration column (Pharmacia) and
collected in 300 .mu.l fractions. TABLE-US-00001 TABLE 1 An example
of the purification of phytase from wheat bran Enzyme Specific
Puri- activity Activity fica- Volume (.mu.molP/ Protein (.mu.molP/
tion Yield Step (ml) min) (mg) min/mg) fold % Sample ready 1000
3500 2230 1.57 1 100 for SP-sepharose 650 2652 674 3.94 2.5 76
SP-sepharose 80 1375 573 2.40 1.5 39 60% (NH.sub.4).sub.2SO.sub.4+
180 530 119 4.46 2.8 24 buffer change 43 151 27.3 5.51 3.5 7
Ultrogel 8 52 0.38 136.80 87.1 2 Ressource S Gel filtration
SDS-PAGE Analysis
[0219] After assaying for phytase activity the proteins in 100
.mu.l of each fraction from the gelfiltration step were
precipitated with methaNOI and chloroform (Wessel and Flugge, 1984)
and subjected to SDS-PAGE using 4-12% Nupage gradient gels (NOVEX)
and a Mes buffer pH 7.4. The gel was electrophoresed according to
the manufactures manual with M12 (NOVEX) as molecular weight
standard. Proteins were detected by silverstaining carried out as
follows. The gel was soaked in 50% methanol, 10% acetic acid for 1
hour and rinsed in water 5 times during 1 hour. It was then
incubated in 5 .mu.g/ml dithiothreitol (DTT) for 30 min., rinsed
quickly with water and soaked in 0.1% aqueous methanol for 1 hour.
The gel was then rinsed once with water and once with 3%
Na.sub.2CO.sub.3 before soaking in developer (100 ml 3%
Na.sub.2CO.sub.3 containing 25 .mu.l 37% formaldehyde). The
reaction was stopped by the addition of 6 g citric acid.
Molecular Weight Determination
[0220] A superose (Pharmacia) gelfiltration column was
equillibrated in 100 mM NaAc, 300 mM Nacl pH 5.5 and the elution
time for the molecular weight standards IgG, Albumin, carbonic
anhydrase, cytochrome C and Vitamin B12 (all from Pharmacia)
determined. The concentrated semi-purified phytase was loaded in
200 .mu.l portions and the eluent was collected in 200 .mu.l
fractions.
Determination of pH Optimum
[0221] Substrate solutions containing 5 mM phytate were prepared at
different pH (4.0, 4.6, 5.0, 5.5, 6.0 NaAc; 6.0, 6.5, 7.0 Bis-Tris;
6.8, 7.6, 8.1 Tes buffer), and incubated with the purified wheat
bran phytase for 30 min at 37.degree. C.
Determination of Temperature Optimum
[0222] The assay was carried out at 25, 37, 45, 50, 55, and
60.degree. C., with the assay described above by incubating for 30
min at the indicated temperature Lineweaver-Burk plot was made from
the results of incubating the wheat bran phytate substrate
solutions of descending concentrations that were prepared in 50 mM
NaAc. Substrate solutions with the following concentrations 5;
1.667; 1; 0.714; 0.556; 0.385; 0.294 mM were made and pH adjusted
to 5.5. Wheat bran phytase has a broad temperature optimum (see
FIG. 4). The reactions were incubated in a thermoblock at the
indicated temperature for 30 min. The highest value of activity
(reached at 45.degree. C.) was set to 100%.
Determination of the Effect of Divalent Cation on Phytase
Activity
[0223] This was investigated by the addition of CaCl.sub.2,
FeSO.sub.4, MgCl, MnSO.sub.4, LiCI,
(NH.sub.4).sub.6Mo.sub.7O.sub.24, to the assay mixture. The final
concentration used was 0.2, 1 and 5 mM. Gelfiltrated sample 5 .mu.l
(20 ng phytase) was added to each tube before incubating for 30 min
at 37.degree. C.
Determination of the Isoelectric Point:
[0224] Proteins were focused at 11.degree. C. for a total of 2.5
kVh in non-denaturing isoelectric-focusing polyacrylamid gels (IEF)
(Ampholine PAGplate pH 3.5-9.5 (Pharmacia)). An IEF calibration kit
(Pharmacia) was applied to determine the pH-gradient across the
gel. The IEF markers were visualised by Coomassie staining
according to the recommendations of the manufacturer. The presence
of acid phosphatase was identified by incubation of the gel at
37.degree. C. in 100 ml 0.05M citrate, pH 4.5 with 50 mg
Na-1-naphtyl phosphate (Merck 6815), 25 mg Farst Garnet GBC (Sigma
F-8761) and 0.5 ml 10% MgCl.
[0225] MDD-HPLC analyses of the degradation products: The substrate
solution (Engelen et al., 1994) was diluted 1:10 and 20 .mu.l used
for assays with 2 .mu.l enzyme and 8 .mu.l 100 mM NaAc, pH 5.5. The
reaction mixture was either stopped immediately or incubated 10 min
at 37.degree. C. The samples were analysed by the HPLC based metal
dye-detection method as described previously (Mayr, 1990).
Amino Acid Sequencing:
[0226] For amino acid sequencing, phytase containing sample was
precipitated with methanol and chloroform prior to SDS-PAGE with
Nupage gel as described above. Proteins were electroblofted onto
0.45ptm PVDF membrane (polyvinylidene diflouride) (Millipore) using
a semidry blofting system (Kyhse-Andersen, 1984) (Hoefer Semiphor,
Pharmacia).
[0227] The membrane was stained with coomassie blue for 30 seconds
to visualise the proteins. Two bands corresponding to a molecular
mass of .about.56 kD were excised and the proteins subjected to
Edman degradation and the N-terminal sequence was determined.
[0228] Highly purified phytase fraction as shown in FIG. 1 lane 3
was sucked onto a filter and cleaved with CNBr in order to obtain
internal amino acid sequence. The products were loaded onto the
sequencer without prior separation.
Specificity of Phytase Antibody:
[0229] Synthetic peptides corresponding to a 12 N-terminal amino
acid sequence of the purified phytase enzyme (see: SEQ ID NO.:7)
were conjugated to the carrier protein Keyhole limpet hemocyanin
(synthesis and conjugation, K. J. Ross-Petersen, Horsholm) and was
used to raise antibodies in a rabbit. The serum from third bleed
was tested in western blots.
Westernblot:
[0230] SDS-PAGE and electroblofting of wheat bran protein samples
was performed and the PVDF membrane was blocked with 2% Tween 20 in
TBS (1.times.TBS: 6.06 g Tris; 8.77 NaCl; pr. litre pH adjusted to
7.4 with HCl.) for 10 min. The membrane was washed in TBS and
incubated in 0.5% Tween 20 in TBS with 1:500 V/V serum containing
the wheat bran phytase antibody for one hour. The membrane was
washed three times 5 min in TBS and incubated with 1:2000
swine-anti-rabbit alkaline phosphatase conjugated secondary
antibodies (DAKO, Denmark) for 1 hour. Incubations were performed
at room temperature in sealed plastic bags. The membrane was then
three times 5 min. in TBS before staining in 20 ml freshly made 0.1
M ethanolamine pH 9.6, with 1.2 mg 5-bromo4-choro-3-indoyl
phosphate (BCIP), 3 mg nitroblue tetrazolium (NBT) and 80 .mu.l 1M
MgCl. The developed blot was washed in water and dried.
MALDI MS sample preparation and analysis:
[0231] Gel slices containing the .about.56 and .about.66 kD were
excised from the NuPage gel, transferred to separate eppendorf tube
and cut in 1 mm.sup.2 pieces. The gel particles were washed with
water and 0.1 M NH.sub.4HCO.sub.3/acetonitrile 1:1 (v/v), all
remaining liquid removed and acetonitrile added to cover the gel
particles. Acetonitrile was removed and the gel particles
rehydrated in 0.1 M NH.sub.4HCO.sub.3. After 5 minutes, an equal
volume of acetonitrile was added and the pieces incubated for 15
min. The liquid was removed and gel particles dried down in a
vacuum centrifuge.
[0232] The protein was reduced and alkylated by first swelling the
gel particles in 10 mM dithiotreitol/0.1 M NH.sub.4HCO.sub.3 and
incubating for 45 min at 56.degree. C. After chilling to room
temperature the liquid was quickly replaced with roughly the same
volume of freshly prepared 55 mM iodoacetamide in 0.1 M
NH.sub.4HCO.sub.3. The protein containing gel particles were
incubated for 30 minutes at room temperature in the dark.
lodoacetamide solution was removed, and the gel particles washed
with 0.1 M NH.sub.4HCO.sub.3 and acetonitrile as described
above.
[0233] Gel particles were completely dried down in a speed vac, and
subsequently rehydrated in a freshly prepared and chilled digestion
buffer containing 50 mM NH.sub.4HCO.sub.3, 5 mM CaCl.sub.2 and 12.5
ng/.mu.L of trypsin (Promega, modified, sequencing grade) at
4.degree. C. The remaining supernatant was, after 45 min, replaced
with 5-20 .mu.L of the same buffer without trypsin. The digestion
reaction was incubated at 37.degree. C. overnight.
[0234] The peptides were extracted from the gel with acetonitrile
containing 25 mM NH.sub.4HCO.sub.3, and then dried in a vacuum
centrifuge. The peptides were re-dissolved in 10 .mu.l 5% formic
acid and 0.5 .mu.l analysed by MALDI MS using the dried droplet
method.
Immuno-Chemistry
[0235] A synthetic peptide conjugated to a carrier protein was used
to raise antibodies in rabbit. As can be seen in FIG. 6, the
polyclonal antibodies clearly mark the 66 kD peptide band at very
low concentrations in both the highly purified and in a
semi-purified wheat bran extract. The 25 kD band visible in lane 5
is due to unspecific interactions with the major impurity (clearly
visible in the coomassie stained lane 3) in this fraction.
[0236] Westernblot: SDS-PAGE and electroblofting of wheat bran
protein samples was performed and the PVDF membrane was blocked
with 2% Tween 20 in TBS (1.times.TBS: 6.06 g Tris; 8.77 NaCl; pr.
litre pH adjusted to 7.4 with HCl.) for 10 min. The membrane was
washed in TBS and incubated in 0.5% Tween 20 in TBS with 1:500 VN
serum containing the wheat bran phytase antibody for one hour. The
membrane was washed three times 5 min in TBS and incubated with
1:2000 swine-anti-rabbit alkaline phosphatase conjugated secondary
antibodies (DAKO, Denmark) for 1 hour. Incubations were performed
at room temperature in sealed plastic bags. The membrane was then
three times 5 min. in TBS before staining in 20 ml freshly made 0.1
M ethanolamine pH 9.6, with 1.2 mg 5-bromo-4-choro-3-indoyl
phosphate (BCIP), 3 mg nitroblue tetrazolium (NBT) and 80 .mu.l 1M
MgCl. The developed blot was washed in water and dried.
Cloning
[0237] The following is an example of cloning and sequencing
procedures for identification of nucleotide sequences encoding
phytase enzymes from wheat and barley:
PCR Primers and Oligonucleotides
[0238] The following oligonucleotides were used in the isolation of
the wheat phytase gene and cDNA: TABLE-US-00002 Name Sequence INT
F1 V1 TAYCCIGGICGIATIGCIGTIGTIGGIGA INT F1 V2
TAYCCIGGIAGRATIGCIGTIGTIGGIGA HVR1 GAGCGCTCGTACGCGTGCACATGGCC HVR2
TTCCCGCCGTCGCCCACCGAGAT HVR3 GGGCAGTGCCCCGGCTCGTCGGC HVR4
GAGATGTACACGGCGCCGCA HVR5 GGTCCTGGTTCCTGTGCCATC PH-W-1
ACGTCTTGTTGCCGATCTGCTCCTC PH-W-2 AGGAGCAGGAGTAGCAGTCCGTTCC PH-W-3
GAGGTGGTGTTGTACGTGAG Ad1 GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCC
CGGGCTGGT Ad2 ACCAGCCC AP1 GTAATACGACTCACTATAGGGC AP2
ACTATAGGGCACGCGTGGT PH-W2-1 AAAGAATCGGCGGCGAGCCCGTAGCG PH-W2-2
CGTGCCGGGGTCCAGCGGCTTGACGG PH-W2-3 ACGGTGCCGCCCATCTGG PH-RT-F
ACGTACAACACCACCTCGACCG PH-RT-R CGGGTCCAGCGTGTAGTTGAAC PH-cDNA-F2
ACACTCACCTCGCACTGCTCTC PH-cDNA-R TTACGGACCGTGTGCGGGCCTGGTCCAGTT
Purification of DNA and RNA
[0239] Chromosomal DNA (gDNA) was isolated from leaf material from
young seedlings of wheat (cv. Kadett) or barley (cv. Alexis) by
homogenisation in liquid N.sub.2 followed by several
phenol/chloroform extractions and ethanol precipitation (Sharp et
al., 1988). Total RNA was isolated from developing wheat kernels
harvested approximately 20 days after anthesis using TRIzol reagent
(Life Technologies) according to the manufacturers
instructions.
Cloning and Sequencing
[0240] PCR products were cloned into the pCR4-TOPO vector using
"TOPO TA Cloning Kit for Sequencing" (Invitrogen) according to the
manufacturers instructions. Sequencing reactions with T3, T7 or
gene insert specific primers were performed using the DYEnamic ET
sequencing kit (Amersham Pharmacia Biotech) according to
instructions, separated on a Perkin Elmer ABI Prism 377 automated
sequencer and analysed using the accompanying software, Sequencher
v3.1.1 (Gene Codes Corporation) and MacVector v7.0 (Oxford
Molecular) software packages. A minimum of three independent clones
of each PCR product were sequenced to resolve PCR based
mis-incorporations.
Genomic PCR Using Degenerate Primers
[0241] PCR reactions (50 mM KCl; 10 mM Tris-HCl, pH 9.0; 0.1%
Triton X-100; 1.25 mM MgCl.sub.2; 0.2 mM dNTPs; 0.2 u Taq
polymerase) with 100 ng wheat gDNA, 20 pmol of a 1:1 mix of two
degenerate forward primers (INT F1 V1, INT F1 V2) corresponding to
the reverse translation of fhe internal phytase peptide
(YPGRIAVVGD) and 5 pmol of a reverse primer derived from the barley
EST clone BE602374 (HVR1, HVR2, HVR3, HVR4 or HVR5) with the
following parameters: 35.times.(95.degree. C., 0 m 45 s; 60.degree.
C. 1 m 00; 72.degree. C. 2 m 30 s). The PCR product obtained with
the HVR3 reverse primer were re-amplified using 1/40 of the
original PCR reaction as template, the same forward primer and the
internal HVR2 reverse primer, confirming that it represented a
phytase sequence. The re-amplification product was gel-purified
using the Geneclean Spin Kit (BIO101), cloned and sequenced. The
resulting sequence covers 728 bp of coding sequence interrupted by
two introns of approximately 100 bp each, at conserved positions
relative to the introns in the putative purple acid phosphatase
from Arabidopsis (AAF20233).
Genomic Walk PCR
[0242] To identify the 5' end of the wheat phytase gene, genomic
walk libraries were prepared by digestion of wheat gDNA with a
restriction enzyme giving blunt-ended fragments (EcoRV, DraI, PvuII
or HindII) followed by ligation of an asymmetric adapter (Ad1+Ad2)
in which Taq polymerase extension of the short lower strand is
blocked by an amino-modification of the 3' end. Primary PCR
reactions were performed using walk library corresponding to 6.25
ng gDNA as template and the first adapter primer (AP1) in
combination with PH-W-1 (annealing 300 bp from the 5' end of the
sequence obtained by degenerate PCR) using polymerase mix and
reagents from the "Expand Long Template PCR kit" (Boehringer
Mannheim) and the following parameters: 7.times.(94.degree. C. 0 m
45 s, 72.degree. C. 3 m 30 s)+32.times.(94.degree. C. 0 m 45 s,
67.degree. C. 3 m 45 s, 67.degree. C. 3 m 30 s). Secondary nested
PCR reactions were performed under identical conditions and cycling
parameters using the nested adapter primer (AP2), PH-W-2 (annealing
151 bp upstream of PH-W-1) and 1/40 of the primary PCR reaction as
template. A tertiary PCR reaction was performed using AP2, PH-W-3
(annealing 263 bp upstream of PH-W-1) and 1/40 of the secondary PCR
reaction as template with parameters: 25.times.(94.degree. C. 0 m
45 s, 50.degree. C. 0 m 45 s, 72.degree. C. 2 m 30 s). A specific
PCR product amplified from the HindII library was isolated, cloned
and sequenced. Although this extended the phytase coding sequence
with 322 bp upstream of the 5' end of the degenerate PCR product,
covering a 97 bp intron again at a conserved position, the
amino-terminal peptide sequence was not included on this fragment.
New walk primers (PH-W2-1, PH-W2-2, PH-W2-3) were constructed based
on the additional sequence information and identical nested PCR
reactions were repeated on the original walk libraries. Cloning and
sequencing of a 541 bp fragment amplified from the PvuII library
revealed that it covered a fourth conserved intron, the amino
terminal peptide sequence and included the 5' end of the coding
sequence.
RT-PCR
[0243] Primers for RT-PCR amplification of the wheat phytase coding
sequence (PH-cDNA-F2, PH-RT-F, PH-RT-R and PH-cDNA-R) were designed
from the identified genomic sequence. First strand cDNA synthesis
was performed using total RNA from developing kernels using
Superscriptll (GIBCO-BRL) reverse transcriptase with either of the
two reverse primers under the conditions described by the
manufacturer: Amplification of the entire cDNA using the
PH-cDNA-F2/PH-cDNA-R primer combination was not possible probably
due to a very high G+C content (>80%) particularly in the first
part of the coding region and the relatively large size of the
product. Alternative the cDNA was isolated as two overlapping
fragments using the primer combinations PH-cDNA-F2/PH-RT-R and
PH-RT-F/PH-cDNA-R under the following conditions 95.degree. C. 1 m
30 s, 35.times.(95.degree. C. 0 m 30 s, 65.degree. C. 1 m 45 s,
72.degree. C. 2 m 00 s).
Characteristics of the Wheat Phytase cDNA Sequence
[0244] The wheat phytase cDNA sequence contains an open reading
frame of 1620 nucleotides encoding a deduced amino acid sequence
540 amino acids.
[0245] The wheat phytase is predicted (using the signalP prediction
server at http://www.cbs.dtu.dk; Nielsen et al., 1997) to contain a
signal peptide targeting it for export through the endoplasmic
reticulum, possibly with the vacuole as the final destination. The
signal peptide cleavage site is predicted to be immediately
preceding one of the four amino acids from position 19-22, which is
in excellent agreement with the amino terminal sequence found in
the purified enzyme. Assuming signal peptide cleavage after
position 21 of the deduced amino acid sequence, the predicted
molecular mass of the mature phytase polypeptide is 57.7 kDa in
excellent correspondence with the estimated molecular mass of 56
kDa for the purified phytase enzyme. Analysis of the deduced
protein sequence reveals the presence of 8 putative N-glycosylation
sites. One or more of these sites might be used for modification of
the protein during transport through the endoplasmic reticulum and
golgi apparatus, resulting in the creation of a phytase population
with heterogenous mass. Thus, glycosylation might explain the
detection of phytase activities of higher apparent molecular
masses.
[0246] Scanning the deduced phytase sequence against the Prosite
(Hofmann et al., 1999) and Pfam (Bateman et al., 1999) databases
resulted in only one significant hit: the Pfam purple acid
phosphatase profile, strongly suggesting that the wheat phytase
belongs to this enzyme class. Additionally, alignment of the
deduced wheat phytase sequence with kidney bean purple acid
phosphatase (P80366) reveals that all seven metal-chelating and
several neighbouring residues, which have been identified in the
active site of the lafter by X-ray crystallography are conserved in
the wheat phytase.
[0247] Seven of the tryptic fragments predicted from the deduced
wheat phytase sequence correspond closely in molecular mass to
fragments identified in MALDI-TOF MS analysis of trypsin treated
purified wheat bran phytase (FIG. 7). The matched fragments, five
of which are found in both the 56 and 66 kDa bands of the wheat
bran phytase and two of which overlaps strongly conserved sites in
purple acid phosphatase, are the following: TABLE-US-00003 Position
predicted MW Observed MW 380-404 2859.07 2858.38 351-369 2263.48
2263.11 432-447 1734.79 1736.87 43-54 1292.30 1292.63 484-495
1302.35 1305.74 246-256 1328.39 1328.68 107-116 1064.14 1064.58
[0248] This confirms the relation between the purified wheat bran
phytase and the cloned cDNA sequence. However, the presence of
multiple unmatched fragments together with the three amino acid
differences between the sequences identified by Edman degradation
of the purified phytase and the sequence deduced from the cDNA
strongly suggest that they represent products from two different
genes. This is not unexpected considering that common domesticated
wheat is hexaploid and thus probably harbouring phytase genes, that
might have diverged, on each of the three constituent genomes.
Sequence CWU 1
1
51 1 15 PRT Triticum aestivum 1 Glu Pro Ala Ser Thr Leu Thr Gly Pro
Ser Arg Pro Val Thr Val 1 5 10 15 2 19 PRT Triticum aestivum
MISC_FEATURE (2)..(2) Xaa = any amino acid 2 Met Xaa Ala Val Gly
Ser Asp Ser Tyr Pro Gly Arg Ile Ala Val Val 1 5 10 15 Gly Asp Leu 3
15 PRT Triticum aestivum MISC_FEATURE (3)..(4) Xaa = Any amino acid
MISC_FEATURE (6)..(6) Xaa = Any amino acid MISC_FEATURE (9)..(10)
Xaa = Any amino acid MISC_FEATURE (12)..(13) Xaa = Any amino acid 3
Met Leu Xaa Xaa Tyr Xaa Asp Tyr Xaa Xaa Ser Xaa Xaa Gln Tyr 1 5 10
15 4 20 PRT Triticum aestivum 4 Thr Met Ser Ala Asn Gly Ser Asp Ser
Tyr Pro Gly Arg Ile Ala Val 1 5 10 15 Val Gly Asp Leu 20 5 20 PRT
Triticum aestivum 5 Thr Met Gly Ala Asn Gly Ser Asp Ser Tyr Pro Gly
Arg Ile Ala Val 1 5 10 15 Val Gly Asp Leu 20 6 12 PRT Triticum
aestivum 6 Glu Pro Ala Ser Thr Leu Thr Gly Pro Ser Arg Pro 1 5 10 7
540 PRT Triticum aestivum 7 Met Trp Met Trp Arg Gly Ser Leu Pro Leu
Leu Leu Leu Ala Ala Ala 1 5 10 15 Val Ala Ala Ala Ala Glu Pro Ala
Ser Thr Leu Glu Gly Pro Ser Arg 20 25 30 Pro Val Thr Val Pro Leu
Arg Glu Asp Arg Gly His Ala Val Asp Leu 35 40 45 Pro Asp Thr Asp
Pro Arg Val Gln Arg Arg Val Thr Gly Trp Ala Pro 50 55 60 Glu Gln
Ile Ala Val Ala Leu Ser Ala Ala Pro Thr Ser Ala Trp Val 65 70 75 80
Ser Trp Ile Thr Gly Asp Phe Gln Met Gly Gly Ala Val Lys Pro Leu 85
90 95 Asp Pro Gly Thr Val Gly Ser Val Val Arg Tyr Gly Leu Ala Ala
Asp 100 105 110 Ser Leu Val Arg Glu Ala Thr Gly Asp Ala Leu Val Tyr
Ser Gln Leu 115 120 125 Tyr Pro Phe Glu Gly Leu Gln Asn Tyr Thr Ser
Gly Ile Ile His His 130 135 140 Val Arg Leu Gln Gly Leu Glu Pro Gly
Thr Lys Tyr Tyr Tyr Gln Cys 145 150 155 160 Gly Asp Pro Ala Ile Pro
Gly Ala Met Ser Ala Val His Ala Phe Arg 165 170 175 Thr Met Pro Ala
Val Gly Pro Arg Ser Tyr Pro Gly Arg Ile Ala Val 180 185 190 Val Gly
Asp Leu Gly Leu Thr Tyr Asn Thr Thr Ser Thr Val Asp His 195 200 205
Met Ala Ser Asn Arg Pro Asp Leu Val Leu Leu Val Gly Asp Val Cys 210
215 220 Tyr Ala Asn Met Tyr Leu Thr Asn Gly Thr Gly Ala Asp Cys Tyr
Ser 225 230 235 240 Cys Ala Phe Gly Lys Ser Thr Pro Ile His Glu Thr
Tyr Gln Pro Arg 245 250 255 Trp Asp Tyr Trp Gly Arg Tyr Met Glu Ala
Val Thr Ser Gly Thr Pro 260 265 270 Met Met Val Val Glu Gly Asn His
Glu Ile Glu Glu Gln Ile Gly Asn 275 280 285 Lys Thr Phe Ala Ala Tyr
Arg Ser Arg Phe Ala Phe Pro Ser Thr Glu 290 295 300 Ser Gly Ser Phe
Ser Pro Phe Tyr Tyr Ser Phe Asp Ala Gly Gly Ile 305 310 315 320 His
Phe Leu Met Leu Gly Ala Tyr Ala Asp Tyr Gly Arg Ser Gly Glu 325 330
335 Gln Tyr Arg Trp Leu Glu Lys Asp Leu Ala Lys Val Asp Arg Ser Val
340 345 350 Thr Pro Trp Leu Val Ala Gly Trp His Ala Pro Trp Tyr Thr
Thr Tyr 355 360 365 Lys Ala His Tyr Arg Glu Val Glu Cys Met Arg Val
Ala Met Glu Glu 370 375 380 Leu Leu Tyr Ser His Gly Leu Asp Ile Ala
Phe Thr Gly His Val His 385 390 395 400 Ala Tyr Glu Arg Ser Asn Arg
Val Phe Asn Tyr Thr Leu Asp Pro Cys 405 410 415 Gly Ala Val His Ile
Ser Val Gly Asp Gly Gly Asn Arg Glu Lys Met 420 425 430 Ala Thr Thr
His Ala Asp Glu Pro Gly His Cys Pro Asp Pro Arg Pro 435 440 445 Lys
Pro Asn Ala Phe Ile Gly Gly Phe Cys Ala Phe Asn Phe Thr Ser 450 455
460 Gly Pro Ala Ala Gly Arg Phe Cys Trp Asp Arg Gln Pro Asp Tyr Ser
465 470 475 480 Ala Tyr Arg Glu Ser Ser Phe Gly His Gly Ile Leu Glu
Val Lys Asn 485 490 495 Glu Thr His Ala Leu Trp Arg Trp His Arg Asn
Gln Asp Met Tyr Gly 500 505 510 Ser Ala Gly Asp Glu Ile Tyr Ile Val
Arg Glu Pro His Arg Cys Leu 515 520 525 His Lys His Asn Trp Thr Arg
Pro Ala His Gly Pro 530 535 540 8 1623 DNA Triticum aestivum 8
atgtggatgt ggagggggtc gctgccgctg cttctgctcg ccgcggcggt ggcggcggcg
60 gctgagccgg cgtcgacgct ggagggaccg tcgcggccgg tgacggtgcc
gctgcgggaa 120 gacaggggcc acgcggtgga cctgccggac acggaccccc
gggtgcagcg ccgggtcaca 180 ggctgggctc ccgagcagat cgccgtcgcg
ctctccgccg ctcccacctc cgcctgggtc 240 tcctggatca caggggattt
ccagatgggc ggcgccgtca agccgctgga ccccggcacg 300 gtcggcagcg
tcgtgcgcta cggcctcgcc gccgattctt tggtccgcga ggccaccggc 360
gacgcgctcg tgtacagcca gctctacccc ttcgagggcc tccagaacta cacctccggc
420 atcatccacc acgtccgcct ccaagggctt gagcctggga cgaagtacta
ctaccagtgc 480 ggcgacccgg ccatcccggg ggcgatgagc gccgtccacg
cgttccggac gatgccggcg 540 gtcgggccgc ggagctaccc ggggaggatc
gccgtggtgg gggacctcgg gctcacgtac 600 aacaccacct cgaccgtgga
ccacatggcg agcaaccggc cggacctggt cctcctcgtc 660 ggcgacgtgt
gctacgccaa catgtacctc accaacggca ccggagcgga ctgctactcg 720
tgcgcgttcg gcaagtcgac gcccatccac gagacgtacc agccgcgctg ggactactgg
780 ggaaggtaca tggaggcggt gacgtcgggg acgccgatga tggtggtgga
agggaaccat 840 gagatagagg agcagatcgg gaacaagacg ttcgcggcct
accgctcccg gttcgcgttc 900 ccgtcgacgg agagcgggtc cttctccccc
ttctactact cgttcgacgc cggcgggatc 960 catttcctca tgctcggcgc
ctacgccgac tacggcaggt caggggagca gtacagatgg 1020 ctggagaagg
acctggcgaa ggtggacagg tcggtgacgc cgtggctggt cgccggctgg 1080
cacgcgccat ggtacaccac ctacaaggct cactacaggg aggtggagtg catgagagtg
1140 gccatggagg agctgctcta ctcccacggc ctcgacatcg ccttcaccgg
ccatgtgcac 1200 gcgtatgagc gctccaaccg ggtgttcaac tacacgctgg
acccgtgcgg cgccgtgcac 1260 atctcggtgg gcgacggcgg gaaccgcgag
aagatggcca ccacccacgc cgacgagcca 1320 gggcactgcc cggacccgcg
gcccaagccc aacgccttca tcggcggctt ctgcgccttt 1380 aacttcacgt
ccggcccggc cgccggcagg ttctgctggg accggcagcc ggactacagc 1440
gcctaccggg agagcagctt cggccacggc atcctcgagg tgaagaacga gacgcacgct
1500 ctgtggagat ggcacaggaa ccaggacatg tacgggagcg ccggagatga
gatttacatt 1560 gtccgggagc cgcacaggtg cttgcacaaa cacaactgga
ccaggcccgc acacggtccg 1620 taa 1623 9 455 PRT Hordeum vulgare 9 Glu
Phe Gln Met Gly Gly Thr Val Lys Pro Leu Asp Pro Arg Thr Val 1 5 10
15 Gly Ser Val Val Arg Tyr Gly Leu Ala Ala Asp Ser Leu Val Arg Glu
20 25 30 Ala Thr Gly Asp Ala Leu Val Tyr Ser Gln Leu Tyr Pro Phe
Glu Gly 35 40 45 Leu His Asn Tyr Thr Ser Gly Ile Ile His His Val
Arg Leu Gln Gly 50 55 60 Leu Glu Pro Gly Thr Lys Tyr Tyr Tyr Gln
Cys Gly Asp Pro Ala Ile 65 70 75 80 Pro Gly Ala Met Ser Ala Val His
Ala Phe Arg Thr Met Pro Ala Ala 85 90 95 Gly Pro Arg Ser Tyr Pro
Gly Arg Ile Ala Val Val Gly Asp Leu Gly 100 105 110 Leu Thr Tyr Asn
Thr Thr Ser Thr Val Asp His Met Thr Ser Asn Arg 115 120 125 Pro Asp
Leu Val Val Leu Val Gly Asp Val Ser Tyr Ala Asn Met Tyr 130 135 140
Leu Thr Asn Gly Thr Gly Thr Asp Cys Tyr Ser Cys Ser Phe Gly Lys 145
150 155 160 Ser Thr Pro Ile His Glu Thr Tyr Gln Pro Arg Trp Asp Tyr
Trp Gly 165 170 175 Arg Tyr Met Glu Pro Val Thr Ser Ser Thr Pro Met
Met Val Val Glu 180 185 190 Gly Asn His Glu Ile Glu Glu Gln Ile Gly
Asn Lys Thr Phe Ala Ala 195 200 205 Tyr Arg Ser Arg Phe Ala Phe Pro
Ser Ala Glu Ser Gly Ser Phe Ser 210 215 220 Pro Phe Tyr Tyr Ser Phe
Asp Ala Gly Gly Ile His Phe Ile Met Leu 225 230 235 240 Gly Ala Tyr
Ala Asp Tyr Gly Arg Ser Gly Glu Gln Tyr Arg Trp Leu 245 250 255 Glu
Lys Asp Leu Ala Lys Val Asp Arg Ser Val Thr Pro Trp Leu Val 260 265
270 Ala Gly Trp His Ala Pro Trp Tyr Ala Thr Tyr Lys Ala His Tyr Arg
275 280 285 Glu Val Glu Cys Met Arg Val Ala Met Glu Glu Leu Leu Tyr
Ser His 290 295 300 Gly Leu Asp Ile Ala Phe Thr Gly His Val His Ala
Tyr Glu Arg Ser 305 310 315 320 Asn Arg Val Phe Asn Tyr Thr Leu Asp
Pro Cys Gly Ala Val Tyr Ile 325 330 335 Ser Val Gly Asp Gly Gly Asn
Arg Glu Lys Met Ala Thr Thr His Ala 340 345 350 Asp Glu Pro Gly His
Cys Pro Asp Pro Arg Pro Lys Pro Asn Ala Phe 355 360 365 Ile Ala Gly
Phe Cys Ala Phe Asn Phe Thr Ser Gly Pro Ala Ala Gly 370 375 380 Arg
Phe Cys Trp Asp Arg Gln Pro Asp Tyr Ser Ala Tyr Arg Glu Ser 385 390
395 400 Ser Phe Gly His Gly Ile Leu Glu Val Lys Asn Glu Thr His Ala
Leu 405 410 415 Trp Arg Trp His Arg Asn Gln Asp Leu Tyr Gly Ser Ala
Gly Asp Glu 420 425 430 Ile Tyr Ile Val Arg Glu Pro Glu Arg Cys Trp
His Lys His Asn Trp 435 440 445 Thr Arg Pro Ala His Gly Pro 450 455
10 1370 DNA Hordeum vulgare 10 gggaattcca gatgggcggc accgtgaagc
cgctggaccc ccgcacggtc ggcagcgtcg 60 tgcgctacgg gctcgccgcc
gactctttgg ttcgcgaggc caccggcgac gcgctcgtgt 120 acagccagct
ctaccccttc gagggcctcc acaactacac ctccggcatc atccaccacg 180
tccgcctcca agggcttgag cctgggacca agtactacta ccagtgcggc gacccggcca
240 tcccgggggc gatgagcgcc gtccacgcgt tccggacgat gccggcggcg
gggccgcgga 300 gctacccggg gaggatcgcc gtggtgggag acctcgggct
cacgtacaac accacctcga 360 ccgtggacca catgacgagc aaccggccgg
acctggtcgt cctcgtcggc gacgtcagct 420 acgccaacat gtacctcacc
aacggcaccg gaacggactg ctactcctgc tccttcggca 480 agtcaacgcc
catccacgaa acctaccagc cgcgctggga ctactgggga aggtacatgg 540
agccggtgac gtcgagcacg ccgatgatgg tggtggaagg gaaccacgag atagaggagc
600 agatcggcaa caagacgttc gcggcctacc gctcccggtt cgcgttcccg
tcggcggaga 660 gcgggtcctt ctcccccttc tactactcct tcgacgccgg
cgggatccac ttcatcatgc 720 tcggcgccta cgccgactac ggcaggtcag
gggagcagta cagatggctg gagaaggacc 780 tggcgaaggt ggacaggtcg
gtgaccccct ggctggtggc cggctggcac gcgccatggt 840 acgccacgta
caaggctcac tacagggagg tggagtgcat gagagtggcc atggaggagc 900
tgctctactc ccacggcctc gacatcgcct tcaccggcca tgtgcacgcg tacgagcgct
960 ccaaccgggt gttcaactac acgctggacc cgtgcggcgc cgtgtacatc
tcggtgggcg 1020 acggcgggaa ccgggagaag atggccacca cccacgccga
cgagccgggg cactgcccgg 1080 acccgcggcc aaagcccaac gccttcattg
ccggcttctg cgcctttaac ttcacgtccg 1140 gcccggccgc cggcaggttc
tgctgggacc ggcagccgga ctacagcgcg taccgggaga 1200 gcagcttcgg
ccatggcatc ctcgaggtga agaacgagac gcacgctctg tggagatggc 1260
acaggaacca ggacctgtac gggagcgccg gagatgagat ttacattgtt cgggagccgg
1320 aaaggtgctg gcacaagcac aactggacca ggcccgcaca cggtccgtaa 1370 11
1220 DNA Hordeum vulgare 11 atgtcgattt ggagggggtc gctgccgctg
tttctgcttc tgctcgcggc ggcgacggct 60 gagccggcgt cgatgctgga
gggcccgtct gggccggtga cggtgctgct gcaggaagac 120 aggggccacg
cggtggacct gccggacacg gacccccggg tgcagcgccg ggtcacaggc 180
tgggctcccg agcagatcgc cgtcgcgctc tccgccgctc ccacctccgc ctgggtctca
240 tggatcacag gggatttcca gatgggcggc gctgtcaagc cgctggaccc
aggcacggtc 300 ggcagcgtcg tgcgctacgg cctcgccgcc gattctgtgg
tccgcgaggc caccggcgac 360 gcgctcgtct acagccagct ctaccccttt
gagggcctcc agaactacac ctccggcatc 420 atccaccacg tccgcctcca
aggtcttgag cctgggacga agtactacta ccagtgcggc 480 gacccggcca
tcccgggggc gatgagcgcc gtccacgcat tccggacgat gccggccgtg 540
gggccgcgga gctacccggg gaggatcgcc gtggtgggag atctcgggct cacgtacaac
600 accacgtcga ccgtggagca catggcgagc aaccagccgg acctggtcct
cctggtcggc 660 gacgtgagct acgccaacct gtacctgacc aacggcacgg
gaacagactg ctactcctgc 720 tcgttcgcca agtccacgcc catccacgag
acgtaccagc cgcgctggga ttactgggga 780 aggtacatgg agcccgtgac
gtcgagcacg ccgatgatgg tggtcgaagg gaaccacgag 840 atcgagcagc
agatcggcaa caagaccttc gcggcttaca gcgcgcggtt cgcgttcccg 900
tcgaaagaga gcgagtcctt ctcccccttc tactactcct tcgacgttgg cggcatccat
960 ttcatcatgc tcgctgccta cgcgaactac agtaaatcag gagaccagta
cagatggttg 1020 gagaaggacc tagcaaaggt ggatagatca gtgaccccat
ggctggtcgc cgggtggcac 1080 gcgccgtggt acagcaccta caaggctcac
tacagggagg cggagtgcat gagagtggcc 1140 atggaggagc tgctctactc
ctacggcatc gacatcgtct tcaccggcca tgtgcacgcg 1200 tacgagcgct
ccaaccgggt 1220 12 406 PRT Hordeum vulgare 12 Met Ser Ile Trp Arg
Gly Ser Leu Pro Leu Phe Leu Leu Leu Leu Ala 1 5 10 15 Ala Ala Thr
Ala Glu Pro Ala Ser Met Leu Glu Gly Pro Ser Gly Pro 20 25 30 Val
Thr Val Leu Leu Gln Glu Asp Arg Gly His Ala Val Asp Leu Pro 35 40
45 Asp Thr Asp Pro Arg Val Gln Arg Arg Val Thr Gly Trp Ala Pro Glu
50 55 60 Gln Ile Ala Val Ala Leu Ser Ala Ala Pro Thr Ser Ala Trp
Val Ser 65 70 75 80 Trp Ile Thr Gly Asp Phe Gln Met Gly Gly Ala Val
Lys Pro Leu Asp 85 90 95 Pro Gly Thr Val Gly Ser Val Val Arg Tyr
Gly Leu Ala Ala Asp Ser 100 105 110 Val Val Arg Glu Ala Thr Gly Asp
Ala Leu Val Tyr Ser Gln Leu Tyr 115 120 125 Pro Phe Glu Gly Leu Gln
Asn Tyr Thr Ser Gly Ile Ile His His Val 130 135 140 Arg Leu Gln Gly
Leu Glu Pro Gly Thr Lys Tyr Tyr Tyr Gln Cys Gly 145 150 155 160 Asp
Pro Ala Ile Pro Gly Ala Met Ser Ala Val His Ala Phe Arg Thr 165 170
175 Met Pro Ala Val Gly Pro Arg Ser Tyr Pro Gly Arg Ile Ala Val Val
180 185 190 Gly Asp Leu Gly Leu Thr Tyr Asn Thr Thr Ser Thr Val Glu
His Met 195 200 205 Ala Ser Asn Gln Pro Asp Leu Val Leu Leu Val Gly
Asp Val Ser Tyr 210 215 220 Ala Asn Leu Tyr Leu Thr Asn Gly Thr Gly
Thr Asp Cys Tyr Ser Cys 225 230 235 240 Ser Phe Ala Lys Ser Thr Pro
Ile His Glu Thr Tyr Gln Pro Arg Trp 245 250 255 Asp Tyr Trp Gly Arg
Tyr Met Glu Pro Val Thr Ser Ser Thr Pro Met 260 265 270 Met Val Val
Glu Gly Asn His Glu Ile Glu Gln Gln Ile Gly Asn Lys 275 280 285 Thr
Phe Ala Ala Tyr Ser Ala Arg Phe Ala Phe Pro Ser Lys Glu Ser 290 295
300 Glu Ser Phe Ser Pro Phe Tyr Tyr Ser Phe Asp Val Gly Gly Ile His
305 310 315 320 Phe Ile Met Leu Ala Ala Tyr Ala Asn Tyr Ser Lys Ser
Gly Asp Gln 325 330 335 Tyr Arg Trp Leu Glu Lys Asp Leu Ala Lys Val
Asp Arg Ser Val Thr 340 345 350 Pro Trp Leu Val Ala Gly Trp His Ala
Pro Trp Tyr Ser Thr Tyr Lys 355 360 365 Ala His Tyr Arg Glu Ala Glu
Cys Met Arg Val Ala Met Glu Glu Leu 370 375 380 Leu Tyr Ser Tyr Gly
Ile Asp Ile Val Phe Thr Gly His Val His Ala 385 390 395 400 Tyr Glu
Arg Ser Asn Arg 405 13 18 PRT Soybean phytase MISC_FEATURE
(16)..(16) Xaa can be any naturally occurring amino acid 13 Met His
Ala Asp Gln Asp Tyr Cys Ala Asn Pro Gln Lys Tyr Asn Xaa 1 5 10 15
Ala Ile 14 29 PRT Wheat 14 Thr Ala Tyr Cys Cys Ile Gly Gly Ile Cys
Gly Ile Ala Thr Ile Gly 1 5 10 15 Cys Ile Gly Thr Ile Gly Thr Ile
Gly Gly Ile Gly Ala 20 25 15 29 PRT Wheat 15 Thr Ala Tyr Cys Cys
Ile Gly Gly Ile Ala Gly Arg Ala Thr Ile Gly 1 5 10 15 Cys Ile Gly
Thr Ile Gly Thr Ile Gly Gly Ile Gly Ala 20 25 16 26 PRT Wheat 16
Gly Ala Gly Cys Gly Cys Thr Cys Gly Thr Ala Cys Gly Cys Gly Thr 1 5
10 15 Gly Cys Ala Cys Ala Thr Gly Gly Cys Cys 20 25 17 23 PRT Wheat
17 Thr Thr Cys Cys Cys Gly Cys Cys Gly Thr Cys Gly Cys Cys Cys Ala
1 5 10 15 Cys Cys Gly Ala Gly Ala Thr 20 18 23 PRT Wheat 18 Gly Gly
Gly Cys Ala Gly Thr Gly Cys Cys Cys Cys Gly Gly Cys Thr 1 5 10
15 Cys Gly Thr Cys Gly Gly Cys 20 19 20 PRT Wheat 19 Gly Ala Gly
Ala Thr Gly Thr Ala Cys Ala Cys Gly Gly Cys Gly Cys 1 5 10 15 Cys
Gly Cys Ala 20 20 21 PRT Wheat 20 Gly Gly Thr Cys Cys Thr Gly Gly
Thr Thr Cys Cys Thr Gly Thr Gly 1 5 10 15 Cys Cys Ala Thr Cys 20 21
25 PRT Wheat 21 Ala Cys Gly Thr Cys Thr Thr Gly Thr Thr Gly Cys Cys
Gly Ala Thr 1 5 10 15 Cys Thr Gly Cys Thr Cys Cys Thr Cys 20 25 22
25 PRT Wheat 22 Ala Gly Gly Ala Gly Cys Ala Gly Gly Ala Gly Thr Ala
Gly Cys Ala 1 5 10 15 Gly Thr Cys Cys Gly Thr Thr Cys Cys 20 25 23
20 PRT Wheat 23 Gly Ala Gly Gly Thr Gly Gly Thr Gly Thr Thr Gly Thr
Ala Cys Gly 1 5 10 15 Thr Gly Ala Gly 20 24 48 PRT Wheat 24 Gly Thr
Ala Ala Thr Ala Cys Gly Ala Cys Thr Cys Ala Cys Thr Ala 1 5 10 15
Thr Ala Gly Gly Gly Cys Ala Cys Gly Cys Gly Thr Gly Gly Thr Cys 20
25 30 Gly Ala Cys Gly Gly Cys Cys Cys Gly Gly Gly Cys Thr Gly Gly
Thr 35 40 45 25 8 PRT Wheat 25 Ala Cys Cys Ala Gly Cys Cys Cys 1 5
26 22 PRT Wheat 26 Gly Thr Ala Ala Thr Ala Cys Gly Ala Cys Thr Cys
Ala Cys Thr Ala 1 5 10 15 Thr Ala Gly Gly Gly Cys 20 27 19 PRT
Wheat 27 Ala Cys Thr Ala Thr Ala Gly Gly Gly Cys Ala Cys Gly Cys
Gly Thr 1 5 10 15 Gly Gly Thr 28 26 PRT Wheat 28 Ala Ala Ala Gly
Ala Ala Thr Cys Gly Gly Cys Gly Gly Cys Gly Ala 1 5 10 15 Gly Cys
Cys Cys Gly Thr Ala Gly Cys Gly 20 25 29 26 PRT Wheat 29 Cys Gly
Thr Gly Cys Cys Gly Gly Gly Gly Thr Cys Cys Ala Gly Cys 1 5 10 15
Gly Gly Cys Thr Thr Gly Ala Cys Gly Gly 20 25 30 18 PRT Wheat 30
Ala Cys Gly Gly Thr Gly Cys Cys Gly Cys Cys Cys Ala Thr Cys Thr 1 5
10 15 Gly Gly 31 22 PRT Wheat 31 Ala Cys Gly Thr Ala Cys Ala Ala
Cys Ala Cys Cys Ala Cys Cys Thr 1 5 10 15 Cys Gly Ala Cys Cys Gly
20 32 22 PRT Wheat 32 Cys Gly Gly Gly Thr Cys Cys Ala Gly Cys Gly
Thr Gly Thr Ala Gly 1 5 10 15 Thr Thr Gly Ala Ala Cys 20 33 22 PRT
Wheat 33 Ala Cys Ala Cys Thr Cys Ala Cys Cys Thr Cys Gly Cys Ala
Cys Thr 1 5 10 15 Gly Cys Thr Cys Thr Cys 20 34 30 PRT Wheat 34 Thr
Thr Ala Cys Gly Gly Ala Cys Cys Gly Thr Gly Thr Gly Cys Gly 1 5 10
15 Gly Gly Cys Cys Thr Gly Gly Thr Cys Cys Ala Gly Thr Thr 20 25 30
35 10 PRT Wheat 35 Tyr Pro Gly Arg Ile Ala Val Val Gly Asp 1 5 10
36 48 PRT Kidney bean 36 Val Val Val Val Ser Asn Gly Gly Lys Ser
Ser Asn Phe Val Arg Lys 1 5 10 15 Thr Asn Lys Asn Arg Asp Met Pro
Leu Asp Ser Asp Val Phe Arg Val 20 25 30 Pro Pro Gly Tyr Asn Ala
Pro Gln Gln Val His Ile Thr Gln Gly Asp 35 40 45 37 54 PRT
Arabisopsis 37 Thr Ile Pro Thr Thr Leu Asp Gly Pro Phe Lys Pro Leu
Thr Arg Arg 1 5 10 15 Phe Glu Pro Ser Leu Arg Arg Gly Ser Asp Asp
Leu Pro Met Asp His 20 25 30 Pro Arg Leu Arg Lys Arg Asn Val Ser
Ser Asp Phe Pro Glu Gln Ile 35 40 45 Ala Leu Ala Leu Ser Thr 50 38
54 PRT Arabidopsis 38 Thr Ile Pro Thr Thr Leu Asp Gly Pro Phe Lys
Pro Leu Thr Arg Arg 1 5 10 15 Phe Glu Pro Ser Leu Arg Arg Gly Ser
Asp Asp Leu Pro Met Asp His 20 25 30 Pro Arg Leu Arg Lys Arg Asn
Val Ser Ser Asp Phe Pro Glu Gln Ile 35 40 45 Ala Leu Ala Leu Ser
Thr 50 39 53 PRT Arabidopsis 39 Ser Ile Pro Ser Thr Leu Asp Gly Pro
Phe Val Pro Val Thr Val Pro 1 5 10 15 Leu Asp Thr Ser Leu Arg Gly
Gln Ala Ile Asp Leu Pro Asp Thr Asp 20 25 30 Pro Arg Val Arg Arg
Arg Val Ile Gly Phe Glu Pro Glu Gln Ile Ser 35 40 45 Leu Ser Leu
Ser Ser 50 40 31 PRT Soybean 40 His Ile Pro Ser Thr Leu Glu Gly Pro
Phe Asp Pro Val Thr Val Pro 1 5 10 15 Phe Asp Pro Ala Leu Arg Gly
Val Ala Val Asp Leu Pro Glu Thr 20 25 30 41 15 PRT Wheat 41 Glu Pro
Ala Ser Tyr Leu Thr Gly Pro Ser Arg Pro Val Thr Val 1 5 10 15 42 19
PRT Kidney bean 42 Thr Pro Pro Gln Thr Gly Leu Asp Val Pro Tyr Thr
Phe Gly Leu Ile 1 5 10 15 Gly Asp Leu 43 20 PRT Arabidopsis 43 Thr
Met Pro Lys Ser Thr Ser Glu Asn Tyr Pro His Arg Ile Val Val 1 5 10
15 Ala Gly Asp Leu 20 44 20 PRT Arabidopsis 44 Thr Leu Pro Leu Pro
Ser Lys Asp Ala Tyr Pro His Arg Ile Ala Phe 1 5 10 15 Val Gly Asp
Leu 20 45 20 PRT Arabidopsis 45 Thr Met Pro Val Ser Ser Pro Ser Ser
Tyr Pro Gly Arg Ile Ala Val 1 5 10 15 Val Gly Asp Leu 20 46 19 PRT
Wheat MISC_FEATURE (2)..(2) Xaa can be any naturally occurring
amino acid 46 Met Xaa Ala Val Gly Ser Asp Ser Tyr Pro Gly Arg Ile
Ala Val Val 1 5 10 15 Gly Asp Leu 47 15 PRT Kidney bean 47 Val Leu
Ser Ser Tyr Ser Ala Tyr Gly Arg Gly Thr Pro Gln Tyr 1 5 10 15 48 4
PRT Arabidopsis 48 Ala Asp Gln Tyr 1 49 25 PRT Arabidopsis 49 Met
Leu Gly Ala Tyr Val Asp Tyr Asn Asn Thr Gly Lys Ser Met Asp 1 5 10
15 Thr Leu Glu Val Ser Trp Leu Gln Tyr 20 25 50 15 PRT Arabidopsis
50 Met Leu Gly Ala Tyr Ile Ala Tyr Asp Lys Ser Ala Glu Gln Tyr 1 5
10 15 51 15 PRT Wheat MISC_FEATURE (3)..(4) Xaa can be any
naturally occurring amino acid MISC_FEATURE (6)..(6) Xaa can be any
naturally occurring amino acid MISC_FEATURE (9)..(10) Xaa can be
any naturally occurring amino acid MISC_FEATURE (12)..(13) Xaa can
be any naturally occurring amino acid 51 Met Leu Xaa Xaa Tyr Xaa
Asp Tyr Xaa Xaa Ser Xaa Xaa Gln Tyr 1 5 10 15
* * * * *
References