U.S. patent application number 11/392781 was filed with the patent office on 2006-08-03 for consensus phytases.
This patent application is currently assigned to DSM IP ASSETS B.V.. Invention is credited to Martin Lehmann.
Application Number | 20060172390 11/392781 |
Document ID | / |
Family ID | 8227106 |
Filed Date | 2006-08-03 |
United States Patent
Application |
20060172390 |
Kind Code |
A1 |
Lehmann; Martin |
August 3, 2006 |
Consensus phytases
Abstract
A process for obtaining a consensus protein from a group of
amino acid sequences of a defined protein family, proteins and
polynucleotides so obtained, and compositions containing such
proteins.
Inventors: |
Lehmann; Martin; (Inzlingen,
DE) |
Correspondence
Address: |
Stephen M. Haracz, Esq.;BRYAN CAVE LLP
1290 Avenue of the Americas
New York
NY
10104-3300
US
|
Assignee: |
DSM IP ASSETS B.V.
|
Family ID: |
8227106 |
Appl. No.: |
11/392781 |
Filed: |
March 28, 2006 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10421112 |
Apr 23, 2003 |
7052869 |
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11392781 |
Mar 28, 2006 |
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09634493 |
Aug 8, 2000 |
6579975 |
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10421112 |
Apr 23, 2003 |
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09121425 |
Jul 23, 1998 |
6153418 |
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09634493 |
Aug 8, 2000 |
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Current U.S.
Class: |
435/69.1 ;
435/196; 435/254.2; 435/254.3; 536/23.2 |
Current CPC
Class: |
C07K 14/38 20130101;
A61K 38/00 20130101; C12N 9/16 20130101 |
Class at
Publication: |
435/069.1 ;
435/196; 435/254.3; 435/254.2; 536/023.2 |
International
Class: |
C12N 9/16 20060101
C12N009/16; C12P 21/06 20060101 C12P021/06; C07H 21/04 20060101
C07H021/04; C12N 1/16 20060101 C12N001/16; C12N 1/18 20060101
C12N001/18 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 24, 1997 |
EP |
97112688.3 |
Claims
1-12. (canceled)
13. A consensus protein which has the amino acid sequence selected
from the group consisting of SEQ ID NO:17 and amino acid sequences
containing amino acid additions, deletions, and replacements to SEQ
ID NO:17, which sequences have up to two amino acids which are
different from the sequence of SEQ ID NO:17.
14. A consensus protein which has the amino acid sequence selected
from the group consisting of SEQ ID NO:15 and amino acid sequences
containing amino acid additions, deletions, and replacements to SEQ
ID NO:15, which sequences have up to two amino acids which are
different from the sequence of SEQ ID NO:15.
15. A mutein which has the amino acid sequence of SEQ ID NO:17 with
the proviso that Q at position 50 is replaced by L, T or G.
16. A mutein which has the amino acid sequence of SEQ ID NO:17 with
the proviso that Q at position 50 is replaced by T and Y at
position 51 is replaced by N.
17. A mutein which has the amino acid sequence of SEQ ID NO:17 with
the proviso that Q at position 50 is replaced by L and Y at
position 51 is replaced by N.
24. A polypeptide having the amino acid sequence of SEQ ID
NO:15.
25. A polypeptide having the amino acid sequence of SEQ ID
NO:17.
26. A food composition comprising a foodstuff or feed and an amino
acid sequence selected from the group consisting of SEQ ID NO:15
and amino acid sequences containing amino acid additions,
deletions, and replacements to SEQ ID NO:15 SEQ ID NO:1, which
sequences have up to two amino acids which are different from the
sequence of SEQ ID NO:15.
27. A food composition according to claim 26 comprising a
polypeptide having the amino acid sequence of SEQ ID NO:15.
28. A food composition comprising a foodstuff or feed and an amino
acid sequence selected from the group consisting of SEQ ID NO:17
and amino acid sequences containing amino acid additions,
deletions, and replacements to SEQ ID NO:17, which sequences have
up to two amino acids which are different from the sequence of SEQ
ID NO:17.
29. A food composition according to claim 28 comprising a
polypeptide having the amino acid sequence of SEQ ID NO:17.
Description
BACKGROUND OF THE INVENTION
[0001] Phytases (myo-inositol hexakisphosphate phosphohydrolases;
EC 3.1.3.8) are enzymes that hydrolyze-phytate (myo-inositol
hexakisphosphate) to myo-inositol and inorganic phosphate and are
known to be valuable feed additives.
[0002] A phytase was first described in rice bran in 1907 [Suzuki
et al., Bull. Coll. Agr. Tokio Imp. Univ. 7, 495 (1907)] and
phytases from Aspergillus species in 1911 [ox and Golden, J. Biol.
Chem. 10, 183-186 (1911)]. Phytases have also been found in wheat
bran, plant seeds, animal intestines and in microorganisms [Howsen
and Davis, Enzyme Microb. Technol. 5, 377-382: (1983), Lambrechts
et al., Biotech. Lett. 14, 61-66 (1992), Shieh and Ware, Appl.
Microbiol 16, 1348-1351 (1968)].
[0003] The cloning and expression of the phytase from Aspergillus
niger (ficuum) has been described by Van Hartingsveldt et al., in
Gene, 127, 87-94 (1993) and in European Patent Application,
Publication No. (EP) 420 358 and from Aspergillus niger var.
awamori by Piddington et al., in Gene 133, 55-62 (1993).
[0004] Cloning, expression and purification of phytases with
improved properties have been disclosed in EP 684 313. However,
since there is a still ongoing need for further improved phytases,
especially with respect to their thermostability, it is an object
of the present invention to provide the following process which is,
however, not only applicable to phytases.
SUMMARY OF THE INVENTION
[0005] The invention herein is a process for the preparation of a
consensus protein, especially a phytase. The invention is also
directed to a consensus phytase and to a DNA sequence encoding the
consensus phytase. As is well known, a consensus protein is a new
protein whose sequence is created from sequence information
obtained from at least three other proteins having a similar
biological activity. The object in preparing a consensus protein is
to obtain a single protein which combines the advantageous
properties of the original proteins.
[0006] The process is characterized by the following steps:
a) at least three preferably four amino acid sequences of a defined
protein family are aligned by any standard alignment program known
in the art;
[0007] b) amino acids at the same position according to such
alignment are compared regarding their evolutionary similarity by
any standard program known in the art, whereas the degree of
similarity provided by such a program which defines the least
similarity of the amino acids that is used for the determination of
an amino acid of corresponding positions is set to a less stringent
number and the parameters are set in such a way that it is possible
for the program to determine from only 2 identical amino acids at a
corresponding position an amino acid for the consensus protein;
however, if among the compared amino acid sequences are sequences
that show a much higher degree of similarity to each other than to
the residual sequences, the sequences are represented by their
consensus sequence determined as defined in the same way as in the
present process for the consensus sequence of the consensus protein
or a vote weight of 1 divided by the number of such sequences is
assigned to every of those sequences.
[0008] c) in case no common amino acid at a defined position can be
identified by the program, any of the amino acids of all sequences
used for the comparison, preferably the most frequent amino acid of
all such sequences is selected or an amino acid is selected on the
basis of the consideration given in Example 2.
d) once the consensus sequence has been defined, such sequence is
back-translated into a DNA sequence, preferably using a codon
frequency table of the organism in which expression should take
place;
e) the DNA sequence is synthesized by methods known in the art and
used either integrated into a suitable expression vector or by
itself to transform an appropriate host cell;
f) the transformed host cell is grown under suitable culture
conditions and the consensus protein is isolated from the host cell
or its culture medium by methods known in the art.
[0009] In a preferred embodiment of this process step b) can also
be defined as follows: b) amino acids at the same position
according to such an alignment are compared regarding their
evolutionary similarity by any standard program known in the art,
whereas the degree of similarity provided by such program is set at
the lowest possible value and the amino acid which is the most
similar for at least half of the sequences used for the comparison
is selected for the corresponding position in the amino acid
sequence of the consensus protein.
[0010] Thus the claimed invention is a process for obtaining a
consensus protein from a group of amino acid sequences of a defined
protein family, which comprises:
a) aligning a group consisting of three to one hundred, but
preferably three or four amino acid sequences from a defined
protein family;
[0011] b) comparing the evolutionary similarity of amino acids
which occupy a position in the aligned sequences to select a
consensus amino acid for said position using a system which is so
organized that if two amino acids which occupy said position are
identical, then the identical amino acid is selected as the
consensus amino acid for said position, unless three or more other
amino acids at said position have a higher degree of structural
similarity to each other than to the identical amino acid, in which
case the amino acid which has the highest degree of evolutionary
similarity to the other amino acids is selected as the consensus
amino acid for said position, with the proviso that if a set of
amino acid sequences exists within the group of step a) such that
the amino acid sequences within the set have more evolutionary
similarity to each other than to any of the amino acid sequences of
the group which are not part of the set, then the amino acids which
occupy said position in members of the set will have a vote weight
of one divided by the number of amino acid sequences in the set
where the amino acids which occupy said position in amino acid
sequences which are not in the set will have a vote weight of one,
and repeating the procedure for each position in the aligned group
of amino acid sequences;
c) if no consensus amino acid for said position is obtained by the
method of step b), then any amino acid at said position is selected
as the consensus sequence, preferably the most frequent amino
acid;
d) combining the consensus amino acids obtained in steps b) and c)
obtain a consensus amino acid sequence;
e) translating the consensus amino acid sequence into a DNA
sequence, preferably using a codon frequency table specific to
whichever host organism has been selected for expressing the DNA
sequence;
f) obtaining the DNA sequence and using said DNA sequence to
express a protein which is the consensus protein of the defined
protein family.
[0012] The present invention is also directed to new phytases,
preferably phytases having the amino acid sequence depicted in FIG.
2 and variants and muteins thereof. In addition, the invention
includes polynucleotides which encode such new phytases.
A BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1: Calculation of the consensus phytase sequence from
the alignment of nearly all known fungal phytase amino acid
sequences. The letters represent the amino acid residues in the
one-letter code. The following sequences were used for the
alignment: phyA from Aspergillus terreus 9A-1 (Mitchell et al.,
1997; from amino acid (aa) 27), phyA from Aspergillus terreus
cbs116.46 (van Loon et al., 1997; from aa 27), phyA from
Aspergillus niger var. awamori (Piddington et al., 1993; from aa
27), phyA from Aspergillus niger T213; from aa 27), phyA from
Aspergillus niger strain NRRL3135 (van Hartingsveldt et al., 1993;
from aa 27), phyA from Aspergillus fumigatus ATCC 13073 (Pasamontes
et al., 1997b; from aa, 25), phyA from Aspergillus fumigatus ATCC
32722 (van Loon et al., 1997; from aa 27), phyA from Aspergillus
fumigatus ATCC 58128 (van Loon et al., 1997; from aa 27), phyA from
Aspergillus fumigatus ATCC 26906 (van Loon et al., 1997; from aa
27) phyA from Aspergillus fumigatus ATCC 32239 (van Loon et al.,
1997; from aa 30), phyA from Aspergillus nidulans (Pasamontes et
al., 1997a; from aa 25) phyA from Talaromyces thermophilus
(Pasamontes et al., 1997a; from aa 24), and phyA from
Myceliophthora thermophila (Mitchell et al., 1997; from aa 19). The
alignment was calculated using the program PILEUP. The location of
the gaps was refined by hand. Capitalized amino acid residues in
the alignment at a given position belong to the amino acid
coalition that establish the consensus residue. In bold, beneath
the calculated consensus sequence, the amino acid sequence of the
finally constructed fungal consensus phytase (Fcp) is shown. The
gaps in the calculated consensus sequence were filled by hand
according to principals stated in Example 2.
[0014] FIG. 2: DNA sequence of the fungal consensus phytase gene
(fcp) and of the primers synthesized for gene construction. The
calculated amino acid sequence (FIG. 1) was converted into a DNA
sequence using the program BACKTRANSLATE (Devereux et al., 1984)
and the codon frequency table of highly expressed yeast genes (GCG
program package, 9.0). The signal peptide of the phytase from A.
terreus cbs was fused to the N-terminus. The bold bases represent
the sequences of the oligonucleotides used to generate the gene.
The names of the respective oligonucleotides are noted above or
below the sequence. The underlined bases represent the start and
stop codon of the gene. The bases written in italics show the two
introduced Eco RI sites.
[0015] FIG. 3: Temperature optimum of fungal consensus phytase and
other phytases used to calculate the consensus sequence. For the
determination of the temperature optimum, the phytase standard
assay was performed at a series of temperatures between 37 and
85.degree. C. The phytases used were purified according to Example
5. .gradient., fungal consensus phytase; , A. fumigatus 13073
phytase; .quadrature., A. niger NRRL3135 phytase; .largecircle., A.
nidulans phytase; .box-solid., A. terreus 9A-1 phytase;
.circle-solid., A. terreus cbs phytase.
[0016] FIG. 4: The pH-dependent activity profile of fungal
consensus phytase and of the mutant Q50L, Q50T, and Q50G. The
phytase activity was determined using the standard assay in
appropriate buffers (see Example 9) at different pH-values. Plot a)
shows a comparison of fun gal consensus phytase (.circle-solid.) to
the mutants Q50L (.gradient.), Q50T (), and Q50G (.largecircle.) in
percent activity. Plot b) shows a comparison of fungal consensus
phytase (.largecircle.) to mutant Q50L (.circle-solid.) and Q50T
(.gradient.) using the specific activity of the purified enzymes
expressed in H. polymorpha.
[0017] FIG. 5: The pH-dependent activity profile of the mutants
Q50L, Y51N and Q50T, Y51N in comparison to the mutants Q50T and
Q50L of fungal consensus phytase. The phytase activity was
determined using the standard assay in appropriate buffers (see
Example 9) at different pH-values. Graph a) shows the influence of
the mutation Y51N (.circle-solid.) on mutant Q50L (.largecircle.).
Graph. b) shows the influence of the same mutation (.circle-solid.)
on mutant Q50T (.largecircle.).
[0018] FIG. 6: Substrate specificity of fungal consensus phytase
and its mutants Q50L, Q50T, and Q50G. The bars represent the
relative activity in comparison to the activity with phytic acid
(100%) with a variety of known natural and synthetic phosphorylated
compounds.
[0019] FIG. 7: Differential scanning calorimetry (DSC) of fungal
consensus phytase and its mutant Q50T. The protein samples were
concentrated to ca. 50-60 mg/ml and extensively dialyzed against 10
mM sodium acetate, pH 5.0 A constant heating rate of 10.degree.
C./min was applied up to 90.degree. C. DSC of consensus phytase
Q50T (upper graph) yielded in a melting temperature of 78.9.degree.
C., which is nearly identical to the melting point of fungal
consensus phytase (78.1.degree. C., lower graph).
DETAILED DESCRIPTION OF THE INVENTION
[0020] A preferred embodiment of this whole process can be seen in
a process in which a sequence is choosen from a number of highly
homologous sequences and only those amino acid residues are
replaced which clearly differ from a consensus sequence of this
protein family calculated under moderately stringent conditions,
while at all positions of the alignment where the method is not
able to determine an amino acid under moderately stringent
conditions the amino acids of the preferred sequence are taken.
[0021] It is furthermore an object of the present invention to
provide such a process, wherein the program used for the comparison
of amino acids at a defined position regarding their evolutionary
similarity is the program "PRETTY". It is more specifically an
object of the present invention to provide such a process, wherein
the defined protein family is the family of phytases, especially
wherein the phytases are of fungal origin.
[0022] It is furthermore, an object of the present invention to
provide such processes, wherein the host cell is of eukaryotic,
especially fungal, preferably Aspergillus or yeast, preferably
Saccharomyces or Hansenula origin. It is also an object of the
present invention, to provide a consensus protein obtainable by
such a process. A preferred consensus protein obtained by the
present process is of the defined protein family of phytases. The
especially preferred consensus phytase is created based on phytase
sequences from:
Aspergillus terreus 9A-1, aa 27 (Mitchell et al., 1997);
Aspergillus terreus cbs116.46, aa 27 (van Loon et al., 1997);
Aspergillus niger var. awamori, aa 27 (Piddington et al.,
1993);
Aspergillus niger T213, aa 27;
Aspergillus niger strain NRRL3135, aa 27 (van Hartingsveldt et al.,
1993);
Aspergillus fumigatus ATCC 13073, aa 26 (Pasamontes et al.,
1997);
Aspergillus fumigatus ATCC 32722, aa 26 (van Loon et al.,
1997);
Aspergillus fumigatus ATCC 58128, aa 26 (van Loon et al.,
1997);
Aspergillus fumigatus ATCC 26906, aa 26 (van Loon et al.,
1997);
Aspergillus fumigatus ATCC 32239, aa 30 (van Loon et al.,
1997);
Aspergillus nidulans, aa 25 (Pasamontes et al., 1997a);
Talaromyces thermophilus ATCC 20186, aa 24 (Pasamontes et al.,
1997a); and
Myceliophthora thermophila, aa 19 (Mitchell et al., 1997).
Therefore the preferred group of amino acid sequences used in the
process of this invention is the amino acid sequences encoding the
phytases of the above fungi.
[0023] The preferred phytase of the invention is a consensus
protein whose sequence is created based on the sequences of the
twelve phytases shown in Table 3, below, but which is not highly
homologous to any of the twelve phytases in that the consensus
phytase is not more than about 80% identical to any of the twelve
phytases. The present invention is particularly directed to a
consensus phytase which has the amino acid sequence shown in FIG. 2
or a variant or mutein thereof. The consensus phytase of FIG. 2 is
not highly homologous to any of the twelve phytases which were used
to create its sequence, as can be seen from the sequence comparison
results shown in Table 3. Another consensus phytase of this
invention has the sequence shown in FIG. 1 as consensus phytase
(bottom line in boldface type) or a variant or mutein thereof.
[0024] A "variant" of the consensus phytase with amino acid
sequence shown in FIG. 1 or preferably FIG. 2 is the consensus
phytase of Figure or preferably FIG. 2 in which at one or more
positions amino acids have been deleted, added or replaced by one
or more other amino acids with the proviso that the resulting
sequence provides for a phytase whose basic properties like
enzymatic activity (type of and specific activity),
thermostability, activity in a certain pH-range (pH-stability) have
not significantly been changed. "Significantly" means in this
context that a skilled person would say that the properties of the
variant may still be different but would not be unobvious over the
ones of the consensus phytase with the amino acid sequence of FIG.
1 or FIG. 2 itself.
[0025] A mutein refers in the context of the present invention to
replacements of the amino acid in the amino acid sequence of the
consensus protein shown in FIG. 1 o preferably FIG. 2 which lead to
consensus proteins with further improved properties, e.g.,
activity. Such muteins can be defined and prepared on the basis of
the teachings given in European Patent Application number
97810175.6, e.g., Q50L, Q50T, Q50G, Q50L-Y51N, or Q50T-Y51N. "Q50L"
means in this context that at position 50 of the amino acid
sequence the amino acid Q has been replaced by amino acid L.
Therefore specific muteins of this invention include a mutein which
has the amino acid sequence of FIG. 2 except that Q at position 50
has been replaced by L, T, or G, and two muteins which have the
amino acid sequence of FIG. 1 except that Q at position 50 has been
replaced by T or L and Y at position 51 has been replaced by N.
[0026] Polynucleotides which encode the consensus phytase of this
invention, i.e., a phytase with the amino acid sequence of FIG. 1
or preferably FIG. 2 or variants and muteins thereof, especially
the specific muteins listed above, are also part of this invention.
Such polynucleotides may be obtained by known methods, for example
by back translation of the mutein's amino acid sequence and PCR
synthesis of the corresponding polynucleotide as described
below.
[0027] In addition, a food, feed, premix or pharmaceutical
composition comprising a consensus protein as defined above is also
an object of the present invention. Food, feed, and premix
compositions, preferably for domestic livestock, are well known to
a skilled person, as are pharmaceutical compositions. Such
pharmaceutical compositions are likely to be veterinary
compositions formulated for oral ingestion, such as pills and the
like.
[0028] In this context "at least three preferably four amino acid
sequences of such defined protein family" means that three, four,
five, six to 12, 20, 50, 100 or even more sequences can be used for
the alignment and the comparison to create the amino acid sequence
of the consensus protein. Amino acid sequences may be obtained from
known sources such as publications or databases, or may be deduced
by translation of DNA sequences which are publicly available, or
may be determined by known techniques for sequencing an isolated
protein or obtaining and sequencing a gene encoding a protein and
translating the DNA sequence. "Sequences of a defined protein
family" means that such sequences fold into a three dimensional
structure, wherein the .alpha.-helixes, the .beta.-sheets and-turns
are at the same position so that such structures are, as called by
the skilled person, superimposable. Furthermore these sequences
characterize proteins which show the same type of biological
activity, e.g., a defined enzyme class such as the phytases. As
known in the art, the three dimensional structure of one of such
sequences is sufficient to allow the modelling of the structure of
the other sequences of such a family. An example, how this can be
effected, is given in the Reference Example of the present
case.
[0029] Aligning amino acid sequences is a well known process
whereby two or more amino acids are lined up in such a way to
maximize the internal amino acid sequences which they have in
common.
[0030] "Evolutionary similarity" in the context of the present
invention refers to a schema which classifies amino acids regarding
their structural similarity which allows that one amino acid can be
replaced by another amino acid with a minimal influence on the
overall structure, as this is done e.g. by programs, like "PRETTY",
known in the art. The phrase "the degree of similarity provided by
such a program . . . is set to less stringent number" means in the
context of the present invention that values for the parameters
which determine the degree of similarity in the program used in the
practice of the present invention are chosen in a way to allow the
program to define a common amino acid for a maximum of positions of
the whole amino acid sequence, e.g. in case of the program PRETTY a
value of 2 or 3 for the THRESHOLD and a value of 2 for the
PLURALITY can be choosen.
[0031] A consensus amino acid is an amino acid chosen to occupy a
given position in the consensus protein obtained by this method. A
system which is organized to select consensus amino acids as
described above may be a computer program, or a combination of one
or more computer programs with "by hand" analysis and calculation.
A set of amino acid sequences existing within the group of amino
acid sequences from which the consensus sequence is prepared means
a set of such sequences which are more similar to each other than
to other members of the group, based on the evolutionary similarity
analysis performed above. An example of such a group is a species
where a set with in the group would be members of a particular
strain. Furthermore, "a vote weight of one divided by the number of
such sequence means in the context of the present invention that
the sequences which define a group of sequences with a higher
degree of similarity as the other sequences used for the
determination of the consensus sequence only contribute to such
determination with a factor which is equal to one divided by a
number of all sequences of this group. Thus an amino acid occupying
a particular position in the aligned sequences will, if it is a
member of a set, not have a comparison value of equal weight with
the other amino acids (e.g. one) but will have a lower weight
depending on the size of the set which it is in, as the weight is
one divided by the number of amino acid sequences in the set.
[0032] When a consensus amino acid is obtained for each position of
the aligned amino acid sequences, then these consensus amino acids
are "lined up" to obtain the amino acid sequence of the consensus
protein.
[0033] As mentioned before should the program not allow selection
of the most similar amino acid, the most frequent amino acid is
selected, should the latter be impossible the skilled person will
select an amino acid from all the sequences used for the comparison
which is known in the art for its property to improve the
thermostability in proteins as discussed, e.g., by: [0034] Janecek,
S. (1993), Process Biochem. 28, 435-445 or [0035] Fersht, A. R.
& Serrano, L. (1993), Curr. Opin. Struct. Biol. 3, 75-83.
[0036] Alber, T. (1989), Annu. Rev. Biochem. 58, 765-798 or [0037]
Matthews, B. W. (1987), Biochemistry 26, 6885-6888. [0038]
Matthews, B. W. (1991), Curr. Opin. Struct. Biol. 1, 17-21.
[0039] The stability of an enzyme is a critical factor for many
industrial applications. Therefore, a lot of attempts, more or less
successful, have been made to improve the stability, preferably the
thermostability, of enzymes by rational (van den Burg et al., 1998)
or irrational approaches (Akanuma et al. 1998). The forces
influencing the thermostability of a protein are the same those
that are responsible for the proper folding of a peptide strand
(hydrophobic interactions, van der Waals interactions, H-bonds,
salt bridges, conformational strain (Matthews, 1993). Furthermore,
as shown by Matthews et al. (1987), the free energy of the unfolded
state has also an influence on the stability of a protein.
Enhancing of protein stability means to increase the number and
strength of favorable interactions and to decrease the number and
strength of unfavorable interactions. It has been possible to
introduce disulfide linkages (Sauer et al., 1986) to replace
glycine with alanine residues or to increase the proline content in
order to reduce the free energy of the unfolded state (Margarit et
al., 1992; Matthews, 1987a). Other groups concentrated on the
importance of additional H-bonds or salt bridges for the stability
of a protein (Blaber et al., 1993) or tried to fill cavities in the
protein interior to increase the buried hydrophobic surface area
and the van der Waals interactions (Karpusas et al., 1989).
Furthermore, the stabilization of secondary structure elements,
especially .alpha.-helices, for example, by improved helix capping,
was also investigated (Munoz & Serrano, 1995).
[0040] However, there is no fast and promising strategy to identify
amino acid replacements which will increase the stability,
preferably the thermal stability of a protein. Commonly, the 3D
structure of a protein is required to find locations in the
molecule where an amino acid replacement possibly will stabilize
the protein's folded state. Alternative ways to circumvent this
problem are either to search for a homologous protein in a thermo-
or hyperthermophile organism or to detect stability-increasing
amino acid replacements by a random mutagenesis approach. This
latter possibility succeeds in only 10.sup.3 to 10.sup.4 mutations
and is restricted to enzymes for which fast screening procedure is
available (Arase et al., 1993; Risse et al., 1992). For all these
approaches, success was variable and unpredictable and, if
successful, the thermostability enhancements nearly always were
rather small.
[0041] Here we present an alternative way to improve the
thermostability of a protein. Imanaka et al. (1986) were among the
first to use the comparisons of homologous proteins to enhance the
stability of a protein. They used a comparison of proteases from
thermophilic with homologous ones of mesophilic organisms to
enhance the stability of a mesophilic protease. Serrano et al.
(1993) used the comparison of the amino acid sequences of two
homologous mesophilic RNases to construct a more thermostable
Rnase. They mutated individually all of the residues that differ
between the two and combined the mutations that increase the
stability in a multiple mutant. Pantoliano et al. (1989) and, in
particular, Steipe et al. (1994) suggested that the most frequent
amino acid at every position of an alignment of homologous proteins
contribute to the largest amount to the stability of a protein.
Steipe et al. (1994) proved this for a variable domain of an
immunoglobulin, whereas Pantoliano et al. (1989) looked for
positions in the primary sequence of subtilisin in which the
sequence of the enzyme chosen to be improved for higher stability
was singularly divergent. Their approach resulted in the
replacement M50F which increased the T.sub.m of subtilisin by
1.8.degree. C.
[0042] Steipe et al. (1994) proved on a variable domain of
immunoglobulin that it is possible to predict a stabilizing
mutation with better than 60% success rate just by using a
statistical method which determines the most frequent amino acid
residue at a certain position of this domain. It was also suggested
that this method would provide useful results not only for
stabilization of variable domains of antibodies but also for
domains of other proteins. However, it was never mentioned that
this method could be extended to the entire protein. Furthermore,
nothing is said about the program which was used to calculate the
frequency of amino acid residues a distinct position or whether
scoring matrices were used as in the present case.
[0043] It is therefore an object of the present invention to
provide a process for the preparation of a consensus protein
comprising a process to calculate an amino acid residue for nearly
all positions of a so-called consensus protein and to synthesize a
complete gene from this sequence that could be expressed in a pro-
or eukaryotic expression system.
[0044] DNA sequences from which amino acid sequences may be derived
for making consensus proteins of the present invention, can be
constructed starting from genomic or cDNA sequences coding for
proteins, e.g. phytases known in the state of the art [for sequence
information see references mentioned above, e.g. EP-684 313 or
sequence data bases, for example like Genbank (Intelligenetics,
California, USA), European Bioinformatics Institute (Hinston Hall,
Cambridge, GB), NBRF (Georgetown University, Medical Centre,
Washington D.C., USA) and Vecbase (University of Wisconsin,
Biotechnology Centre, Madison, Wis., USA) or disclosed in the
figures by methods of in vitro mutagenesis [see e.g. Sambrook et
al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New
York]. A widely used strategy for such "site directed mutagenesis",
as originally outlined by Hurchinson and Edgell [J. Virol. 8, 181
(1971)], involves the annealing of a synthetic oligonucleotide
carrying the desired nucleotide substitution to a target region of
a single-stranded DNA sequence wherein the mutation should be
introduced [for review see Smith, Annu. Rev. Genet. 19, 423 (1985)
and for improved methods see references 2-6 in Stanssen et al.
Nucl. Acid Res., 17, 4441-4454 (1989)].
[0045] Another possibility of mutating a given DNA sequence which
is also preferred for the practice of the present invention is the
mutagenesis by using the polymerase chain reaction (PCR). DNA as
starting material can be isolated by methods known in the art and
described e.g. in Sambrook et al. (Molecular Cloning) from the
respective strains. For strain information see, e.g., EP 684 313 or
any depository authority indicated below. Aspergillus niger [ATCC
9142], Myceliophthora thermophila [ATCC 48102], Talaromyces
thermophilus [ATCC 20186] and Aspergillus fumigatus [ATCC 34625]
have been redeposited according to the conditions of the Budapest
Treaty at the American Type Culture Cell Collection under the
following accession numbers: ATCC 74337, ATCC 74340, ATCC 74338 and
ATCC 74339, respectively. Amino acid sequences may be obtained by
know methods from these DNA sequences for use in the process of
this invention to obtain a consensus protein. It is however,
understood that DNA encoding a consensus protein in accordance with
the present invention can also be prepared in a synthetic manner as
described, e.g. in EP 747 483 or the examples by methods known in
the art.
[0046] Once complete DNA sequences of the present invention have
been obtained (for example by synthesis based on backtranslation of
a consensus protein obtained in accordance with the invention) they
can be integrated into vectors by methods known in the art and
described e.g. in Sambrook et al. (s.a.) to overexpress the encoded
polypeptide in appropriate host systems. However, a skilled person
knows that also the DNA sequences themselves can be used to
transform the suitable host systems of the invention to get
overexpression of the encoded polypeptide. Appropriate host systems
are for example fungi, like Aspergilli, e.g. Aspergillus niger
[ATCC 9142] or Aspergillus ficuum [NRRL 3135] or like Trichoderma,
e.g. Trichoderma rees or yeasts, like Saccharomyces, e.g.
Saccharomyces cerevisiae or Pichia, like Pichia pastoris, or
Hansenula polymorpha, e.g. H. polymorpha (DSM5215) or plants, as
described, e.g. by Pen et al., Bio/Technology 11, 811-814 (1994). A
skilled person knows that such microorganisms are available from
depository authorities, e.g. the American Type Culture Collection
(ATCC), the Centraalbureau voor Schimmelcultures (CBS) or the
Deutsche Sammlung fur Mikroorganismen und Zellkulturen GmbH (DSM)
or any other depository authority as listed in the Journal
"Industrial Property" [(1991) 1, pages 29-40]. Bacteria which can
be used are e.g. E. coli, Bacilli as, e.g. Bacillus subtilis or
Streptomyces, e.g. Streptomyces lividans (see e.g. Anne and
Mallaert in FEMS Microbiol. Letters 114, 121 (1993). E. coli, which
could be used are E. coli K12 strains e.g. M15 [described as DZ 291
by Villarejo et al. in J. Bacteriol. 120, 466-474 (1974)], HB 101
[ATCC No. 33694]or E. coli SG13009 [Gottesman et al., J. Bacteriol.
148, 265-273 (1981)].
[0047] Vectors which can be used for expression in fungi are known
in the art and described e.g. in EP 420 358, or by Cullen et al.
[Bio/Technology 5, 369-376 (1987)] or Ward in Molecular Industrial
Mycology, Systems and Applications for Filamentous Fungi, Marcel
Dekker, New York (1991), Upshall et al. [Bio/Technology 5,
1301-1304 (1987)] Gwynne et al. [Bio/Technology 5, 71-79 (1987)],
Punt et al. [J. Biotechnol. 17, 19-34 (1991)] and for yeast by
Sreekrishna et al. [J. Basic Microbiol. 28, 265-278 (1988),
Biochemistry 28, 4117-4125 (1989)], Hitzemann et al. [Nature 293,
717-722 (1981)] or in EP-183 070, EP 183 071, EP 248 227, EP 263
311. Suitable vectors which can be used for expression in E. coli
are mentioned, e.g. by Sambrook et al. [s.a.] or by Fiers et al. in
Procd. 8th Int. Biotechnology Symposium" [Soc. Franc. de
Microbiol., Paris (Durand et al., eds.), pp. 680-697 (1988)] or by
Bujard et al. in Methods in Enzymology, eds. Wu and Grossmann,
Academic Press, Inc. Vol. 155, 416-433 (1987) and Stuber et al. in
Immunological Methods, eds. Lefkovits and Pernis, Academic Press,
Inc., Vol. IV, 121-152 (1990). Vectors which could be used for
expression in Bacilli are known in the art and described, e.g. in
EP 405 370, Procd. Natl. Acad. Sci. USA 81, 439 (1984) by Yansura
and Henner, Meth. Enzymol. 185, 199-228 (1990) or EP 207 459.
Vectors which can be used for the expression in H. polymorpha are
known in the art and described, e.g. in Gellissen et al.,
Biotechnology 9, 291-295 (1991).
[0048] Either such vectors already carry regulatory elements, e.g.,
promotors, or the DNA sequences of the present invention can be
engineered to contain such elements. Suitable promotor elements
which can be used are known in the art and are, e.g. for
Trichoderma reesei the cbh1-[Haarki et al., Biotechnology 7,
596-600 (1989)] or the pki1-promotor [Schindler et al.,. Gene 130,
271-275 (1993)], for Aspergillus oryzae the amy-promotor
[Christensen et al., Abstr. 19th Lunteren Lectures on Molecular
Genetics F23 (1987), Christensen et al., Biotechnology 6, 1419-1422
(1988), Tada et al., Mol. Gen. Genet. 229, 301 (1991)], for
Aspergillus niger the glaA-[Cullen et al., Bio/Technology 5,
369-376 (1987), Gwynne et al., Bio/Technology 5, 713-719 (1987),
Ward in Molecular Industrial Mycology, Systems and Applications for
Filamentous Fungi, Marcel Dekker, New York, 83-106 (1991)],
alcA--[Gwynne et al., Bio/Technology 5, 718-719 (1987)],
suc1--[Boddy et al., Curr. Genet. 24, 60-66 (1993)], aphA--[MacRae
et al., Gene 71, 339-348 (1988), MacRae et al., Gene 132, 193-198
(1993)], tpiA--[McKnight et al., Cell 46, 143-147 (1986), Upshall
et al., Bio/Technology 5, 1301-1304 (1987)], gpdA--[Punt et al.,
Gene 69, 49-57 (1988), Punt et al., J. Biotechnol. 17, 19-37
(1991)] and the pkiA-promotor [de Graaff et al., Curr. Genet. 22,
21-27 (1992)]. Suitable promotor elements which could be used for
expression in yeast are known in the art and are, e.g. the
pho5-promotor Vogel et al., Mol. Cell. Biol., 2050-2057 (1989);
Rudolf and Hinnen, Proc. Natl. Acad. Sci. 84, 1340-1344 (1987)] or
the gap-promotor for expression in Saccharomyces cerevisiae and for
Pichia pastoris, e.g. the aox1-promotor [Koutz et al., Yeast 5,
167-177 (1989); Sreekrishna et al., J. Basic Microbiol. 28, 265-278
(1988)], or the FMD promoter [Hollenberg et al., EPA No. 0299108]
or MOX-promotor [Ledeboer et al., Nucleic Acids Res. 13, 3063-3082
(1985)] for H. polymorpha.
[0049] Accordingly vectors comprising DNA sequences of the present
invention, preferably for the expression of said DNA sequences in
bacteria or a fungal or a yeast host and such transformed bacteria
or fungal or yeast hosts are also an object of the present
invention.
[0050] It is also an object of the present invention to provide a
system which allows for high expression of proteins, preferably
phytases like the consensus phytase of the present invention in
Hansenula characterized therein that the codons of the encoding DNA
sequence of such a protein have been selected on the basis of a
codon frequency table of the organism used for expression, e.g.
yeast as in the present case (see e.g. in Example 3) and optionally
the codons for the signal sequence have been selected in a manner
as described for the specific case in Example 3. That means that a
codon frequency table is prepared on the basis of the codons used
in the DNA sequences which encode the amino acid sequences of the
defined protein family. Then the codons for the design of the DNA
sequence of the signal sequence are selected from a codon frequency
table of the host cell used for expression whereby always codons of
comparable frequency in both tables are used.
[0051] Once such DNA sequences have been expressed in an
appropriate host cell in a suitable medium the encoded protein can
be isolated either from the medium in the case the protein is
secreted into the medium or from the host organism in case such
protein is present intracellularly by methods known in the art of
protein purification or described in case of a phytase, e.g. in EP
420 358: Accordingly a process for the preparation of a consensus
protein (i.e. a polypeptide) of the present invention characterized
in that transformed bacteria or a host cell as described above is
cultured under suitable culture conditions and the consensus
protein is recovered therefrom and a consensus protein produced by
such a process or a consensus protein encoded by a DNA sequence of
the present invention are also an object of the present
invention.
[0052] Once obtained, the consensus proteins (i.e. polypeptides),
preferably phytases, of the present invention can be characterized
regarding their properties which make them useful in agriculture.
Any assay known in the art may be used such as those described,
e.g., by Simons et al. [Br. J. Nutr. 64, 525-540 (1990)], Schoner
et al. [J. Anim. Physiol. a. Anim. Nutr. 66, 248-255 (1991)], Vogt
[Arch. Geflugelk. 56, 93-98 (1992)], Jongbloed et al. [J. Anim.
Sci., 70, 1159-1168 (1992)], Perney et al. [Poultry Sci. 72,
2106-2114 (1993)], Farrell et al., [J. Anim. Physiol. a. Anim.
Nutr. 69, 278-283 (1993), Broz et al., [Br. Poultry Sci. 35,
273-280 (1994)] and Dungelhoef et al. [Animal Feed Sci. Technol.
49, 1-10 (1994)].
[0053] In general the consensus phytases of the present invention
can be used without being limited to a specific field of
application, e.g., in case of phytases for the conversion of
inositol polyphosphates, like phytate to inositol and inorganic
phosphate.
[0054] Furthermore the consensus phytases of the present invention
can be used in a process for the preparation of a pharmaceutical
composition or compound food or feeds wherein the components of
such a composition are mixed with one or more consensus phytases of
the present invention. Accordingly compound food or feeds or
pharmaceutical compositions comprising one or more consensus
phytases of the present invention are also an object of the present
invention. A skilled person is familiar with their process of
preparation. Such pharmaceutical compositions or compound foods or
feeds can further comprise additives or components generally used
for such purpose and known in the state of the art.
[0055] It is furthermore an object of the present invention to
provide a process for the reduction of levels of phytate in animal
manure characterized in that an animal is fed such a feed
composition in an amount effective in converting phytate contained
in the feedstuff to inositol and inorganic phosphate.
[0056] The Examples which follow further elucidate the invention
but are not intended to limit it in any way.
EXAMPLES
Reference Example
Homology Modeling of A. fumigatus and A. terreus cbs116.46
Phytase
[0057] The amino acid sequences of A. fumigatus and A. terreus
cbs116.46 phytase were compared with the sequence of A. niger NRRL
3135 phytase (see FIG. 1) for which the three-dimensional structure
had been determined by X-ray crystallography.
[0058] A multiple amino acid sequence alignment of A. niger NRRL
3135 phytase, A. fumigatus phytase and A. terreus cbs116.46 phytase
was calculated with the program "PILEUP" (Prog. Menu for the
Wisconsin Package, version 8, September 1994, Genetics Computer
Group, 575 Science Drive, Madison Wis., USA 53711). The
three-dimensional models of A. fumigatus phytase and A. terreus
cbs116.46 phytase were built by using the structure of A. niger
NRRL 3135 phytase as template and exchanging the amino acids of A.
niger NRRL 3135 phytase according to the sequence alignment to
amino acids of A. fumigatus and A. terreus cbs116.46 phytases,
respectively. Model construction and energy optimization were
performed by using the program Moloc (Gerber and Muller, 1995).
C-alpha positions were kept fixed except for new
insertions/deletions and in loop regions distant from the active
site.
[0059] Only small differences of the modelled structures to the
original crystal structure could be observed in external loops.
Furthermore the different substrate molecules that mainly occur on
the degradation pathway of phytic acid
(myo-inositol-hexakisphosphate) by Pseudomonas sp. bacterium
phytase and, as far as determined, by A. niger NRRL 3135 phytase
(Cosgrove, 1980) were constructed and forged into the active site
cavity of each phytase structure. Each of these substrates was
oriented in a hypothetical binding mode proposed for histidine acid
phosphatases (Van Etten, 1982). The scissile phosphate group was
oriented towards the catalytically essential His 59 to form the
covalent phosphoenzyme intermediate. The oxygen of the substrate
phosphoester bond which will be protonated by Asp 339 after
cleavage was orientated towards the proton donor. Conformational
relaxation of the remaining structural part of the substrates as
well as the surrounding active site residues was performed by
energy optimization with the program Moloc.
[0060] Based on the structure models the residues pointing into the
active site cavity were identified. More than half (60%) of these
positions were identical between these three phytases, whereas only
few positions were not conserved (see FIG. 1). This observation
could be extended to four additional phytase sequences (A.
nidulans, A. terreus 9A1, Talaromyces thermophilus, Myceliophthora
thermophila).
Example 1
Alignment of the Amino Acid Sequence of the Fungal Phytases
[0061] The alignment was calculated using the program PILEUP from
the Sequence Analysis Package Release 9.0 (Devereux et al., 1984)
with the standard parameter (gap creation penalty 12, gap extension
penalty 4). The location of the gaps was refined using a text
editor. Amino acid sequences encoded by the following genes (see
FIG. 1) without the signal sequence were used for the performance
of the alignment starting with the amino acid (aa) mentioned
below:
phyA gene from Aspergillus terreus 9A-1, aa 27 (Mitchell et al.,
1997)
phyA gene from Aspergillus terreus cbs116.46, aa 27 (van Loon et
al., 1997)
phyA gene from Aspergillus niger var. awamori, aa 27 (Piddington et
al., 1993)
phyA gene from Aspergillus niger T213, aa 27
phyA gene from Aspergillus niger strain NRRL3135, aa 27 (van
Hartingsveldt et al., 1993)
phyA gene from Aspergillus fumigatus ATCC 13073, aa 26 (Pasamontes
et al., 1997)
phyA gene from Aspergillus fumigatus ATCC 32722, aa 26 (van Loon et
al., 1997)
phyA gene from Aspergillus fumigatus ATCC 58128, aa 26 (van Loon et
al., 1997)
phyA gene from Aspergillus fumigatus ATCC 26906, aa 26 (van Loon et
al., 1997)
phyA gene from Aspergillus fumigatus ATCC 32239, aa 30 (van Loon et
al., 1997)
phyA gene from Aspergillus nidulans, aa 25 (Pasamontes et al.,
1997a)
phyA gene from Talaromyces thermophilus ATCC 20186, aa 24
(Pasamontes et al., 1997a)
phyA gene from Myceliophthora thermophila, aa 19 (Mitchell et al.,
1997)
[0062] Table 2 shows the homology of the phytase sequences
mentioned above. TABLE-US-00001 TABLE 2 Table 2: Homology of the
fungal phytases. The amino acid sequences of the phytases used in
the alignment were compared by the program GAP (GCG program
package, 9; Devereux et al., 1984) using the standard parameters.
The comparison was restricted to the part of the sequence that was
also used for the alignment (see legend to FIG. 1) lacking the
signal peptide which was rather divergent. The numbers above and
beneath the diagonal represent the amino acid identities and
similarities, respectively. A. niger A. terreus A. terreus NRRL A.
fumigatus 9A-1 cbs116.46 3135 13073 A. nidulans T. thermophilus M.
thermophila % identity A. terreus 89.1 62.0 60.6 59.3 58.3 48.6
9A-1 A. terreus 90.7 63.6 62.0 61.2 59.7 49.1 cbs A. niger 67.3
68.9 66.8 64.2 62.5 49.4 NRRL 3135 A. fumigatus 66.1 67.2 71.1 68.0
62.6 53.0 13073 A. nidulans 65.0 66.7 69.0 73.3 60.5 52.5 T.
thermophilus 63.8 64.5 68.9 68.1 67.4 49.8 M. thermophila 53.7 54.6
57.6 61.0 59.9 57.8 % similarity
Example 2
Calculation of the Amino Acid Sequence of Fungal Consensus
Phytases
[0063] Using the refined alignment of Example 1 as input, the
consensus sequence was calculated by the program PRETTY from the
Sequence Analysis Package Release 9.0 (Devereux et al., 1984).
PRETTY prints sequences with their columns aligned and can display
a consensus sequence for the alignment. A vote weight that pays
regard to the similarity between the amino acid sequences of the
phytases aligned were assigned to all sequences. The vote weight
was set such as the combined impact of all phytases from one
sequence subgroup (same species of origin but different strains),
e.g. the amino acid sequences of all phytases from A. fumigatus, on
the election was set one, that means that each sequence contributes
with a value of 1 divided by the number of strain sequences (see
Table 1). By this means, it was possible to prevent that very
similar amino acid sequences, e.g. of the phytases from different
A. fumigatus strains, dominate the calculated consensus sequence.
TABLE-US-00002 TABLE 1 Table 1: Vote weights of the amino acid
sequences of the fungal phytases used. The table shows the vote
weights used to calculate the consensus sequence of the fungal
phytases. Aspergillus terreus 9A-1 phytase: 0.50 Aspergillus
terreus cbs116.46 phytase: 0.50 Aspergillus niger var. awamori
phytase: 0.3333 Aspergillus niger T213 phytase: 0.3333 Aspergillus
niger NRRL3135 phytase: 0.3333 Aspergillus fumigatus ATCC 13073
phytase: 0.20 Aspergillus fumigatus ATCC 32722 phytase: 0.20
Aspergillus fumigatus ATCC 58128 phytase: 0.20 Aspergillus
fumigatus ATCC 26906 phytase: 0.20 Aspergillus fumigatus ATCC 32239
phytase: 0.20 Aspergillus nidulans phytase: 1.00 Talaromyces
thermophilus ATCC 20186 phytase: 1.00 Myceliophthora thermophila
phytase: 1.00
[0064] The program PRETTY was started with the following
parameters: The plurality defining the number of votes below which
there is no consensus was set on 2.0. The threshold, which
determines the scoring matrix value below which an amino acid
residue may not vote for a coalition of residues, was set on 2.
PRETTY used the PrettyPep.Cmp consensus scoring matrix for
peptides.
[0065] Ten positions of the alignment (position 46, 66, 82, 138,
162, 236, 276, 279, 280, 308; FIG. 1), for which the program was
not able to determine a consensus residue, were filled by hand
according to the following rules: if a most frequent residue
existed, this residue was chosen (138, 236, 280); if a prevalent
group of chemically similar or equivalent residues occurred, the
most frequent or, if not available, one residues of this group was
selected (46, 66, 82, 162, 276, 308). If there was either a
prevalent residue nor a prevalent group, one of the occurring
residues was chosen according to common assumption on their
influence on the protein stability (279). Eight other positions
(132, 170, 204, 211, 275, 317, 384, 447; FIG. 1) were not filled
with the amino acid residue selected by the program but normally
with amino acids that occur with the same frequency as the residues
that were chosen by the program. In most cases, the slight
underrating of the three A. niger sequences (sum of the vote
weights: 0.99) was eliminated by this corrections.
[0066] Table 3 shows the homology of the calculated fungal
consensus phytase amino acid sequence to the phytase sequences used
for the calculation. TABLE-US-00003 TABLE 3 Table 3: Homology of
the amino acid sequence of fungal consensus phytase to the phytases
used for its calculation. The amino acid sequences of all phytases
were compared with the fungal consensus phytase sequence using the
program GAP (GCG program package, 9.0). Again, the comparison was
restricted to that part of the sequence that was used in the
alignment. Phytase Identity [%] Similarity [%] A. niger T213 76.6
79.6 A. niger var. awamori 76.6 79.6 A. niger NRRL3135 76.6 79.4 A.
nidulans 77.4 81.5 A. terreus 9A-1 70.7 74.8 A. terreus cbs116.46
72.1 75.9 A. fumigatus 13073 80.0 83.9 A. fumigatus 32239 78.2 82.3
T. thermophilus 72.7 76.8 M. thermophila 58.3 64.5
Example 3
Conversion of the Fungal Consensus Phytase Amino Acid Sequence to a
DNA Sequence
[0067] The first 26 amino acid residues of A. terreus cbs116.46
phytase were used as signal peptide and, therefore, fused to the
N-terminus of all consensus phytases. For this stretch, we used a
special method to calculate the corresponding DNA sequence. Purvis
et al. (1987) proposed that the incorporation of rare codons in a
gene has an influence on the folding efficiency of the protein.
Therefore, at least the distribution of rare codons in the signal
sequence of A. terreus cbs116.46, which was used for the fungal
consensus phytase and which is very important for secretion of the
protein, but converted into the S. cerevisiae codon usage, was
transferred into the new signal sequence generated for expression
in S. cerevisiae. For the remaining parts of the protein, we used
the codon frequency table of highly expressed S. cerevisiae genes,
obtained from the GCG program package, to translate the calculated
amino acid sequence into a DNA sequence.
The resulting sequence of the fcp gene are shown in FIG. 2.
Example 4
Construction and Cloning of the Fungal Consensus Phytase Genes
[0068] The calculated DNA sequence of fungal consensus phytase was
divided into oligonucleotides of 85 bp, alternately using the
sequence of the sense and the anti-sense strand. Every
oligonucleotide overlaps 20 bp with its previous and its following
oligonucleotide of the opposite strand. The location of all
primers, purchased by Microsynth, Balgach (Switzerland) and
obtained in a PAGE-purified form, is indicated in FIG. 2.
[0069] In three PCR reactions, the synthesized oligonucleotides
were composed to the entire gene. For the PCR, the High Fidelity
Kit from Boehringer Mannheim (Boehringer Mannheim, Mannheim,
Germany) and the thermo cycler The Protokol.TM. from AMS
Biotechnology (Europe) Ltd. (Lugano, Switzerland) were used.
[0070] Oligonucleotide CP-1 to CP-10 (Mix 1, FIG. 2) were mixed to
a concentration of 0.2 pMol/.mu.l per each oligonucleotide. A
second oligonucleotide mixture (Mix 2) was prepared with CP-9 to
CP-22 (0.2 pMol/.mu.l per each oligonucleotide). Additionally, four
short primers were used in the PCR reactions: TABLE-US-00004 CP-a:
EcoRI 5'-TAT ATG AAT TCA TGG GCG TGT TCG TC-3' CP-b: 5'-TGA AAA GTT
CAT TGA AGG TTT C-3' CP-c: 5'-TCT TCG AAA GCA GTA CAA GTA C-3'
CP-e: EcoRI 5'-TAT ATG AAT TCT TAA GCG AAA C-3'
PCR Reaction .alpha.: [0071] 10 .mu.l Mix 1 (2.0 pmol of each
oligonucleotide) [0072] 2 .mu.l nucleotides (10 mM each nucleotide)
[0073] 2 .mu.l primer CP-a (10 pmol/.mu.l) [0074] 2 .mu.l primer
CP-c (10 pmol/.mu.l) [0075] 10.0 .mu.l PCR buffer [0076] 0.75 .mu.l
polymerase mixture [0077] 73.25 .mu.l H.sub.2O PCR Reaction b:
[0078] 10 .mu.l Mix 2 (2.0 pmol of each oligonucleotide) [0079] 2
.mu.l nucleotides (10 mM each nucleotide) [0080] 2 .mu.l primer
CP-b (10 pmol/.mu.l) [0081] 2 .mu.l primer CP-e (10 pmol/.mu.l)
[0082] 10.0 .mu.l PCR buffer [0083] 0.75 .mu.l polymerase mixture
(2.6 U) [0084] 73.25 .mu.l H.sub.2O
[0085] Reaction Conditions for PCR Reaction a and b: TABLE-US-00005
step 1 2 min - 45.degree. C. step 2 30 sec - 72.degree. C. step 3
30 sec - 94.degree. C. step 4 30 sec - 52.degree. C. step 5 1 min -
72.degree. C.
Step 3 to 5 were repeated 40-times.
[0086] The PCR products (670 and 905 bp) were purified by an
agarose gel electrophoresis (0.9% agarose) and a following gel
extraction (QIAEX II Gel Extraction Kit, Qiagen, Hilden, Germany).
The purified DNA fragments were used for the PCR reaction c.
PCR Reaction c:
[0087] 6 .mu.l PCR product of reaction a (.apprxeq.50 ng) [0088] 6
.mu.l PCR product of reaction b (.apprxeq.50 ng) 2 .mu.l primer
CP-a (10 pmol/.mu.l) [0089] 2 .mu.l primer CP-e (10 pmol/.mu.l)
[0090] 10.0 .mu.l PCR buffer [0091] 0.75 .mu.l polymerase mixture
(2.6 U) [0092] 73.25 .mu.l H.sub.2O
[0093] Reaction Conditions for PCR Reaction c: TABLE-US-00006 step
1 2 min - 94.degree. C. step 2 30 sec - 94.degree. C. step 3 30 sec
- 55.degree. C. step 4 1 min - 72.degree. C.
Step 2 to 4 were repeated 31-times.
[0094] The resulting PCR product (1.4 kb) was purified as mentioned
above, digested with Eco RI, and ligated in an Eco RI-digested and
dephosphorylated pBsk(-)-vector (Stratagene, La Jolla, Calif.,
USA). 1 .mu.l of the ligation mixture was used to transform E. coli
XL-1 component cells (Stratagene, La Jolla, Calif., USA). All
standard procedures were carried out as described by Sambrook et
al. (1987). The constructed fungal consensus phytase gene (fcp) was
verified by sequencing (plasmid pBsk.sup.--fcp).
Example 5
Expression of the Fungal Consensus Phytase Gene fcp and its
Variants in Saccharomyces cerevisiae and Their Purification from
Culture Supernatant
[0095] A fungal consensus phytase gene was isolated from the
plasmid pBsk-fcp ligated into the Eco RI sites of the expression
cassette of the Saccharomyces cerevisiae expression vector pYES2
(Invitrogen, San Diego, Calif., USA) or subcloned between the
shortened GAPFL (glyceraldhyde-3phosphate dehydrogenase) promoter
and the pho5 terminator as described by Janes et al. (1990). The
correct orientation of the gene was checked by PCR. Transformation
of S. cerevisiae strains. e.g. INVSc1 (Invitrogen, San Diego,
Calif., USA) was done according to Hinnen et al. (1978). Single
colonies harboring the phytase gene under the control of the GAPFL
promoter were picked and cultivated in 5 ml selection medium
(SD-uracil, Sherman et al., 1986) at 30.degree. C. under vigorous
shaking (250 rpm) for one day. The preculture was then added to 500
ml YPD medium (Sherman et al., 1986) and grown under the same
conditions. Induction of the gall promoter was done according to
manufacturer's instruction. After four days of incubation cell
broth was centrifuged (7000 rpm, GS3 rotor, 15 min, 5.degree. C.)
to remove the cells and the supernatant was concentrated by way of
ultrafiltration in Amicon 8400 cells (PM30 membranes) and
ultrafree-15 centrifugal filter devices (Biomax-30K, Millipore,
Bedford, Mass., USA). The concentrate (10 ml) was desalted on a 40
ml Sephadex G25 Superfine column (Pharmacia Biotech, Freiburg;
Germany), with 10 mM sodium acetate, pH 5.0, serving as elution
buffer. The desalted sample was brought to 2 M
(NH.sub.4).sub.2SO.sub.4 and directly loaded onto a 1 ml Butyl
Sepharose 4 Fast Flow hydrophobic interaction chromatography column
(Pharmacia Biotech, Feiburg; Germany) which was eluted with a
linear gradient from 2 M to 0 M (NH.sub.4).sub.2SO.sub.4 in 10 mM
sodium acetate, pH 5.0. Phytase was eluted in the break-through,
concentrated and loaded on a 120 ml Sephacryl S-300 gel permeation
chromatography column (Pharmacia Biotech, Freiburg, Germany).
Fungal consensus phytase and fungal consensus phytase 7 eluted as a
homogeneous symmetrical peak and was shown by SDS-PAGE to be
approx. 95% pure.
Example 6
Expression of the Fungal Consensus Phytase Genes fcp and its
Variants in Hansenula polymorpha
[0096] The phytase expression vectors, used to transform H.
polymorpha, was constructed by inserting the Eco RI fragment of
pBsk.sup.--fcp encoding the consensus phytase or a variant into the
multiple cloning site of the H. polymorpha expression vector
pFPMT121, which is based on an ura3 selection marker and the FMD
promoter. The 5' end of the fcp gene is fused to the FMD promoter,
the 3' end to the MOX terminator (Gellissen et al., 1996; EP 0299
108 B). The resulting expression vector are designated pFPMTfcp and
pBsk-fcp7.
[0097] The constructed plasmids were propagated in E. coli. Plasmid
DNA was purified using standard state of the art procedures. The
expression plasmids were transformed into the H. polymorpha strain
RP11 deficient in orotidine-5'-phosphate decarboxylase (ura3) using
the procedure for preparation of competent cells and for
transformation of yeast as described in Gelissen et al. (1996).
Each transformation mixture was plated on YNB (0.14% w/v Difco YNB
and 0.5% ammonium sulfate) containing 2% glucose and 1.8% agar and
incubated at 37.degree. C. After 4 to 5 days individual,
transformant colonies were picked and grown in the liquid medium
described above for 2 days at 37.degree. C. Subsequently, an
aliquot of this culture was used to inoculate fresh vials with
YNB-medium containing 2% glucose. After seven further passages in
selective medium, the expression vector integrates into the yeast
genome in multimeric form. Subsequently, mitotically stable
transformants were obtained by two additional cultivation steps in
3 ml non-selective liquid medium (YPD, 2% glucose, 10 g yeast
extract, and 20 g peptone). In order to obtain genetically
homogeneous recombinant strains an aliquot from the last
stabilization culture was plated on a selective plate. Single
colonies were isolated for analysis of phytase expression in YNB
containing 2% glycerol instead of glucose to derepress the fmd
promoter. Purification of the fungal consensus phytases was done as
described in Example 5.
Example 7
Expression of the Fungal Consensus Genes fcp and its Variants in
Aspergillus niger
[0098] Plasmid pBsk-fcp or the corresponding plasmid of a variant
of the fcp gene were used as template for the introduction of a Bsp
HI-site upstream of the start codon of the genes and an Eco RV-site
downstream of the stop codon. The Expand.TM. High Fidelity PCR Kit
(Boehringer Mannheim, Mannheim, Germany) was used with the
following primers: TABLE-US-00007 Primer Asp-1: Bsp HI 5'-TAT ATC
ATG AGC GTG TTC GTC GTG CTA CTG TTC-3' Primer Asp-2 for cloning of
fcp and fcp7: 3'-ACC CGA CTT ACA AAG CGA ATT CTA TAG ATA TAT-5' Eco
RV
[0099] The reaction was performed as described by the supplier. The
PCR-amplified fcp gene had a new Bsp HI site at the start codon,
introduced by primer Asp-1, which resulted in a replacement of the
second amino acid residue glycine by serine. Subsequently, the
DNA-fragment was digested with Bsp HI and Eco RV and ligated into
the Nco I site downstream of the glucoamylase promoter of
Aspergillus niger (glaA) and the Eco RV site upstream of the
Aspergillus nidulans tryptophan C terminator (trpC) (Mullaney et
al., 1985). After this cloning step, the genes were sequenced to
detect possible failures introduced by PCR. The resulting
expression plasmids which basically corresponds to the pGLAC vector
as described in Example 9 of EP 684 313, contained the
orotidine-5'-phosphate decarboxylase gene (pyr4) of Neurospora
crassa as a selection marker. Transformation of Aspergillus niger
and expression of the consensus phytase genes was done as described
in EP 684 313. The fungal consensus phytases were purified as
described in Example 5.
Example 8
Construction of Muteins of Fungal Consensus Phytase
[0100] To construct muteins for expression in A. niger, S.
cerevisiae, or H. polymorpha, the corresponding expression plasmid
containing the fungal consensus phytase gene was used as template
for site-directed mutagenesis. Mutations were introduced using the
"quick exchange.TM. site-directed mutagenesis kit" from Stratagene
(La Jolla, Calif., USA) following the manufacturer's protocol and
using the corresponding primers. All mutations made and the
corresponding primers are summarized in Table 4. Clones harboring
the desired mutation were identified by DNA sequence analysis as
known in the art. The mutated phytase were verified by sequencing
of the complete gene. TABLE-US-00008 TABLE 4 mutation Primer set
Ssp BI Q50L 5'-CAC TTG TGG GGT TTG TAC AGT CCA TAC TTC TC-3' 5'-GAG
AAG TAT GGA CTG TAC AAA CCC CAC AAG TG-3' KpnI Q50T 5'-CAC TTG TGG
GGT ACC TAC TCT CCA TAC TTC TC-3' 5'-GA GAA GTA TGG AGA GTA GGT ACC
CCA CAA GTG-3' Q50G 5'-CAC TTG TGG GGT GGT TAC TCT CCA TAC TTC
TC-3' 5'-GA GAA GTA TGG AGA GTA ACC ACC CCA CAA GTG-3' Kpn I
Q50T-Y51N 5'-CAC TTG TGG GGT ACC AAC TCT CCA TAC TTC TC-3' 5'-GA
GAA GTA TGG AGA GTT GGT ACC CCA CAA GTG-3' Bsa I Q50L-Y51N 5'-CAC
TTG TGG GGT CTC AAC TCT CCA TAC TTC TC-3' 5'-GA GAA GTA TGG AGA GTT
GAG ACC CCA CAA GTG-3' Primers used for the introduction of single
mutations into fungal consensus phytase. For the introduction of
each mutation, two primers containing the desired mutation were
required (see Example 8). The changed triplets are highlighted in
bold letters.
Example 9
Determination of the Phytase Activity and of the Temperature
Optimum of the Consensus Phytase and its Variants
[0101] Phytase activity was determined basically as described by
Mitchell et al. (1997). The activity was measured in a assay
mixture containing 0.5% phytic acid (.apprxeq.5 mM), 200 mM sodium
acetate, pH 5.0. After 15 min incubation at 37.degree. C., the
reaction was stopped by addition of an equal volume of 15%
trichloroacetic acid. The liberated phosphate was quantified by
mixing 100 .mu.l of the assay mixture with 900 .mu.l H.sub.2O and 1
ml Of 0.6 M H.sub.2SO.sub.4, 2% ascorbic acid and 0.5% ammonium
molybdate. Standard solutions of potassium phosphate were used as
reference. One unit of enzyme activity was defined as the amount of
enzyme that releases 1 .mu.mol phosphate per minute at 37.degree.
C. The protein concentration was determined using the enzyme
extinction coefficient at 280 nm calculated according to Pace et
al. (1995): fungal consensus phytase, 1.101; fungal consensus
phytase 7, 1.068. In case of pH-optimum curves, purified enzymes
were diluted in 10 mM sodium acetate, pH 5.0. Incubations were
started by mixing aliquots of the diluted protein with an equal
volume of 1% phytic acid (.apprxeq.10 mM) in a series of different
buffers: 0.4 M glycine/HCl, pH 2.5; 0.4 M acetate/NaOH, pH 3.0,
3.5, 4.0, 4.5, 5.0, 5.5; 0.4 M imidazole/HCl, pH 6.0, 6.5; 0.4 M
Tris/HCl pH 7.0, 7.5, 8.0, 8.5, 9.0. Control experiments showed
that pH was only slightly affected by the mixing step. Incubations
were performed for 15 min at 37.degree. C. as described above.
[0102] For determination of the substrate specificities of the
phytases, phytic acid in the assay mixture was replaced by 5 mM
concentrations of the respective phosphate compounds. The activity
tests were performed as described above.
[0103] For determination of the temperature optimum, enzyme (100
.mu.l) and substrate solution (100 .mu.l) were pre-incubated for 5
min at the given temperature. The reaction was started by addition
of the substrate solution to the enzyme. After 15 min incubation,
the reaction was stopped with trichloroacetic acid and the amount
of phosphate released was determined.
[0104] The pH-optimum of the original fungal consensus phytase was
around pH 6.0-6.5 (70 U/mg). By introduction of the Q50T mutation,
the pH-optimum shifted to pH 6.0 (130 U/mg), while the replacement
by a leucine at the same position resulted in a maximum activity
around pH 5.5 (212 U/mg). The exchange Q50G resulted in a
pH-optimum of the activity above pH 6.0 (see FIG. 4). The exchange
of tyrosine at position 51 with asparagine resulted in a relative
increase of the activity below pH 5.0 (see FIG. 5). Especially by
the Q50L mutation, the specificity for phytate of fungal consensus
phytase was drastically increased (see FIG. 6).
[0105] The temperature optimum of fungal consensus phytase
(70.degree. C.) was 15-25.degree. C. higher than the temperature
optimum of the wild-type phytases (45-55.degree. C.) which were
used to calculate the consensus sequence (see Table 5 and FIG. 3).
TABLE-US-00009 TABLE 5 Table 5: Temperature optimum and
T.sub.m-value of fungal consensus phytase and of the phytases from
A. fumigatus, A. niger, A. nidulans, and M. thermophila. The
temperature optima were taken from FIG. 3. temperature phytase
optimum Tm.sup.a Consensus phytase 70.degree. C. 78.0.degree. C. A.
niger NRRL3135 55.degree. C. 63.3.degree. C. A. fumigatus 13073
55.degree. C. 62.5.degree. C. A. terreus 9A-1 49.degree. C.
57.5.degree. C. A. terreus cbs 45.degree. C. 58.5.degree. C. A.
nidulans 45.degree. C. 55.7.degree. C. M. thermophila 55.degree. C.
.sup.aThe T.sub.m-values were determined by differential scanning
calorimetry as described in Example 10 and shown in FIG. 7.
Example 10
Determination of the Melting Point by Differential Scanning
Calorimetry (DSC)
[0106] In order to determine the unfolding temperature of the
fungal consensus phytases, differential scanning calorimetry was
applied as previously published by Brugger et al. (1997). Solutions
of 50-60 mg/ml homogeneous phytase were used for the tests. A
constant heating rate of 10.degree. C./min was applied up to
90.degree. C.
[0107] The determined melting points clearly show the strongly
improved thermostability of the fungal consensus phytase in
comparison to the wild-type phytases (see Table 5 and FIG. 7). FIG.
7 shows the melting profile of fungal consensus phytase and its
mutant Q50T. Its common melting point was determined between 78 to
79.degree. C.
Sequence CWU 1
1
33 1 440 PRT Aspergillus terreus 1 Lys His Ser Asp Cys Asn Ser Val
Asp His Gly Tyr Gln Cys Phe Pro 1 5 10 15 Glu Leu Ser His Lys Trp
Gly Leu Tyr Ala Pro Tyr Phe Ser Leu Gln 20 25 30 Asp Glu Ser Pro
Phe Pro Leu Asp Val Pro Glu Asp Cys His Ile Thr 35 40 45 Phe Val
Gln Val Leu Ala Arg His Gly Ala Arg Ser Pro Thr His Ser 50 55 60
Lys Thr Lys Ala Tyr Ala Ala Thr Ile Ala Ala Ile Gln Lys Ser Ala 65
70 75 80 Thr Ala Phe Pro Gly Lys Tyr Ala Phe Leu Gln Ser Tyr Asn
Tyr Ser 85 90 95 Leu Asp Ser Glu Glu Leu Thr Pro Phe Gly Arg Asn
Gln Leu Arg Asp 100 105 110 Leu Gly Ala Gln Phe Tyr Glu Arg Tyr Asn
Ala Leu Thr Arg His Ile 115 120 125 Asn Pro Phe Val Arg Ala Thr Asp
Ala Ser Arg Val His Glu Ser Ala 130 135 140 Glu Lys Phe Val Glu Gly
Phe Gln Thr Ala Arg Gln Asp Asp His His 145 150 155 160 Ala Asn Pro
His Gln Pro Ser Pro Arg Val Asp Val Ala Ile Pro Glu 165 170 175 Gly
Ser Ala Tyr Asn Asn Thr Leu Glu His Ser Leu Cys Thr Ala Phe 180 185
190 Glu Ser Ser Thr Val Gly Asp Asp Ala Val Ala Asn Phe Thr Ala Val
195 200 205 Phe Ala Pro Ala Ile Ala Gln Arg Leu Glu Ala Asp Leu Pro
Gly Val 210 215 220 Gln Leu Ser Thr Asp Asp Val Val Asn Leu Met Ala
Met Cys Pro Phe 225 230 235 240 Glu Thr Val Ser Leu Thr Asp Asp Ala
His Thr Leu Ser Pro Phe Cys 245 250 255 Asp Leu Phe Thr Ala Thr Glu
Trp Thr Gln Tyr Asn Tyr Leu Leu Ser 260 265 270 Leu Asp Lys Tyr Tyr
Gly Tyr Gly Gly Gly Asn Pro Leu Gly Pro Val 275 280 285 Gln Gly Val
Gly Trp Ala Asn Glu Leu Met Ala Arg Leu Thr Arg Ala 290 295 300 Pro
Val His Asp His Thr Cys Val Asn Asn Thr Leu Asp Ala Ser Pro 305 310
315 320 Ala Thr Phe Pro Leu Asn Ala Thr Leu Tyr Ala Asp Phe Ser His
Asp 325 330 335 Ser Asn Leu Val Ser Ile Phe Trp Ala Leu Gly Leu Tyr
Asn Gly Thr 340 345 350 Ala Pro Leu Ser Gln Thr Ser Val Glu Ser Val
Ser Gln Thr Asp Gly 355 360 365 Tyr Ala Ala Ala Trp Thr Val Pro Phe
Ala Ala Arg Ala Tyr Val Glu 370 375 380 Met Met Gln Cys Arg Ala Glu
Lys Glu Pro Leu Val Arg Val Leu Val 385 390 395 400 Asn Asp Arg Val
Met Pro Leu His Gly Cys Pro Thr Asp Lys Leu Gly 405 410 415 Arg Cys
Lys Arg Asp Ala Phe Val Ala Gly Leu Ser Phe Ala Gln Ala 420 425 430
Gly Gly Asn Trp Ala Asp Cys Phe 435 440 2 440 PRT Aspergillus
terreus 2 Asn His Ser Asp Cys Thr Ser Val Asp Arg Gly Tyr Gln Cys
Phe Pro 1 5 10 15 Glu Leu Ser His Lys Trp Gly Leu Tyr Ala Pro Tyr
Phe Ser Leu Gln 20 25 30 Asp Glu Ser Pro Phe Pro Leu Asp Val Pro
Asp Asp Cys His Ile Thr 35 40 45 Phe Val Gln Val Leu Ala Arg His
Gly Ala Arg Ser Pro Thr Asp Ser 50 55 60 Lys Thr Lys Ala Tyr Ala
Ala Thr Ile Ala Ala Ile Gln Lys Asn Ala 65 70 75 80 Thr Ala Leu Pro
Gly Lys Tyr Ala Phe Leu Lys Ser Tyr Asn Tyr Ser 85 90 95 Met Gly
Ser Glu Asn Leu Thr Pro Phe Gly Arg Asn Gln Leu Gln Asp 100 105 110
Leu Gly Ala Gln Phe Tyr Arg Arg Tyr Asp Thr Leu Thr Arg His Ile 115
120 125 Asn Pro Phe Val Arg Ala Ala Asp Ser Ser Arg Val His Glu Ser
Ala 130 135 140 Glu Lys Phe Val Glu Gly Phe Gln Asn Ala Arg Gln Gly
Asp Pro His 145 150 155 160 Ala Asn Pro His Gln Pro Ser Pro Arg Val
Asp Val Val Ile Pro Glu 165 170 175 Gly Thr Ala Tyr Asn Asn Thr Leu
Glu His Ser Ile Cys Thr Ala Phe 180 185 190 Glu Ala Ser Thr Val Gly
Asp Ala Ala Ala Asp Asn Phe Thr Ala Val 195 200 205 Phe Ala Pro Ala
Ile Ala Lys Arg Leu Glu Ala Asp Leu Pro Gly Val 210 215 220 Gln Leu
Ser Ala Asp Asp Val Val Asn Leu Met Ala Met Cys Pro Phe 225 230 235
240 Glu Thr Val Ser Leu Thr Asp Asp Ala His Thr Leu Ser Pro Phe Cys
245 250 255 Asp Leu Phe Thr Ala Ala Glu Trp Thr Gln Tyr Asn Tyr Leu
Leu Ser 260 265 270 Leu Asp Lys Tyr Tyr Gly Tyr Gly Gly Gly Asn Pro
Leu Gly Pro Val 275 280 285 Gln Gly Val Gly Trp Ala Asn Glu Leu Ile
Ala Arg Leu Thr Arg Ser 290 295 300 Pro Val His Asp His Thr Cys Val
Asn Asn Thr Leu Asp Ala Asn Pro 305 310 315 320 Ala Thr Phe Pro Leu
Asn Ala Thr Leu Tyr Ala Asp Phe Ser His Asp 325 330 335 Ser Asn Leu
Val Ser Ile Phe Trp Ala Leu Gly Leu Tyr Asn Gly Thr 340 345 350 Lys
Pro Leu Ser Gln Thr Thr Val Glu Asp Ile Thr Arg Thr Asp Gly 355 360
365 Tyr Ala Ala Ala Trp Thr Val Pro Phe Ala Ala Arg Ala Tyr Ile Glu
370 375 380 Met Met Gln Cys Arg Ala Glu Lys Gln Pro Leu Val Arg Val
Leu Val 385 390 395 400 Asn Asp Arg Val Met Pro Leu His Gly Cys Ala
Val Asp Asn Leu Gly 405 410 415 Arg Cys Lys Arg Asp Asp Phe Val Glu
Gly Leu Ser Phe Ala Arg Ala 420 425 430 Gly Gly Asn Trp Ala Glu Cys
Phe 435 440 3 441 PRT Aspergillus niger 3 Asn Gln Ser Thr Cys Asp
Thr Val Asp Gln Gly Tyr Gln Cys Phe Ser 1 5 10 15 Glu Thr Ser His
Leu Trp Gly Gln Tyr Ala Pro Phe Phe Ser Leu Ala 20 25 30 Asn Glu
Ser Ala Ile Ser Pro Asp Val Pro Ala Gly Cys Arg Val Thr 35 40 45
Phe Ala Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Glu Ser 50
55 60 Lys Gly Lys Lys Tyr Ser Ala Leu Ile Glu Glu Ile Gln Gln Asn
Val 65 70 75 80 Thr Thr Phe Asp Gly Lys Tyr Ala Phe Leu Lys Thr Tyr
Asn Tyr Ser 85 90 95 Leu Gly Ala Asp Asp Leu Thr Pro Phe Gly Glu
Gln Glu Leu Val Asn 100 105 110 Ser Gly Ile Lys Phe Tyr Gln Arg Tyr
Glu Ser Leu Thr Arg Asn Ile 115 120 125 Ile Pro Phe Ile Arg Ser Ser
Gly Ser Ser Arg Val Ile Ala Ser Gly 130 135 140 Glu Lys Phe Ile Glu
Gly Phe Gln Ser Thr Lys Leu Lys Asp Pro Arg 145 150 155 160 Ala Gln
Pro Gly Gln Ser Ser Pro Lys Ile Asp Val Val Ile Ser Glu 165 170 175
Ala Ser Ser Ser Asn Asn Thr Leu Asp Pro Gly Thr Cys Thr Val Phe 180
185 190 Glu Asp Ser Glu Leu Ala Asp Thr Val Glu Ala Asn Phe Thr Ala
Thr 195 200 205 Phe Ala Pro Ser Ile Arg Gln Arg Leu Glu Asn Asp Leu
Ser Gly Val 210 215 220 Thr Leu Thr Asp Thr Glu Val Thr Tyr Leu Met
Asp Met Cys Ser Phe 225 230 235 240 Asp Thr Ile Ser Thr Ser Thr Val
Asp Thr Lys Leu Ser Pro Phe Cys 245 250 255 Asp Leu Phe Thr His Asp
Glu Trp Ile His Tyr Asp Tyr Leu Gln Ser 260 265 270 Leu Lys Lys Tyr
Tyr Gly His Gly Ala Gly Asn Pro Leu Gly Pro Thr 275 280 285 Gln Gly
Val Gly Tyr Ala Asn Glu Leu Ile Ala Arg Leu Thr His Ser 290 295 300
Pro Val His Asp Asp Thr Ser Ser Asn His Thr Leu Asp Ser Asn Pro 305
310 315 320 Ala Thr Phe Pro Leu Asn Ser Thr Leu Tyr Ala Asp Phe Ser
His Asp 325 330 335 Asn Gly Ile Ile Ser Ile Leu Phe Ala Leu Gly Leu
Tyr Asn Gly Thr 340 345 350 Lys Pro Leu Ser Thr Thr Thr Val Glu Asn
Ile Thr Gln Thr Asp Gly 355 360 365 Phe Ser Ser Ala Trp Thr Val Pro
Phe Ala Ser Arg Leu Tyr Val Glu 370 375 380 Met Met Gln Cys Gln Ala
Glu Gln Glu Pro Leu Val Arg Val Leu Val 385 390 395 400 Asn Asp Arg
Val Val Pro Leu His Gly Cys Pro Ile Asp Ala Leu Gly 405 410 415 Arg
Cys Thr Arg Asp Ser Phe Val Arg Gly Leu Ser Phe Ala Arg Ser 420 425
430 Gly Gly Asp Trp Ala Glu Cys Ser Ala 435 440 4 441 PRT
Aspergillus niger 4 Asn Gln Ser Ser Cys Asp Thr Val Asp Gln Gly Tyr
Gln Cys Phe Ser 1 5 10 15 Glu Thr Ser His Leu Trp Gly Gln Tyr Ala
Pro Phe Phe Ser Leu Ala 20 25 30 Asn Glu Ser Val Ile Ser Pro Asp
Val Pro Ala Gly Cys Arg Val Thr 35 40 45 Phe Ala Gln Val Leu Ser
Arg His Gly Ala Arg Tyr Pro Thr Glu Ser 50 55 60 Lys Gly Lys Lys
Tyr Ser Ala Leu Ile Glu Glu Ile Gln Gln Asn Val 65 70 75 80 Thr Thr
Phe Asp Gly Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Ser 85 90 95
Leu Gly Ala Asp Asp Leu Thr Pro Phe Gly Glu Gln Glu Leu Val Asn 100
105 110 Ser Gly Ile Lys Phe Tyr Gln Arg Tyr Glu Ser Leu Thr Arg Asn
Ile 115 120 125 Ile Pro Phe Ile Arg Ser Ser Gly Ser Ser Arg Val Ile
Ala Ser Gly 130 135 140 Glu Lys Phe Ile Glu Gly Phe Gln Ser Thr Lys
Leu Lys Asp Pro Arg 145 150 155 160 Ala Gln Pro Gly Gln Ser Ser Pro
Lys Ile Asp Val Val Ile Ser Glu 165 170 175 Ala Ser Ser Ser Asn Asn
Thr Leu Asp Pro Gly Thr Cys Thr Val Phe 180 185 190 Glu Asp Ser Glu
Leu Ala Asp Thr Val Glu Ala Asn Phe Thr Ala Thr 195 200 205 Phe Ala
Pro Ser Ile Arg Gln Arg Leu Glu Asn Asp Leu Ser Gly Val 210 215 220
Thr Leu Thr Asp Thr Glu Val Thr Tyr Leu Met Asp Met Cys Ser Phe 225
230 235 240 Asp Thr Ile Ser Thr Ser Thr Val Asp Thr Lys Leu Ser Pro
Phe Cys 245 250 255 Asp Leu Phe Thr His Asp Glu Trp Ile His Tyr Asp
Tyr Leu Arg Ser 260 265 270 Leu Lys Lys Tyr Tyr Gly His Gly Ala Gly
Asn Pro Leu Gly Pro Thr 275 280 285 Gln Gly Val Gly Tyr Ala Asn Glu
Leu Ile Ala Arg Leu Thr His Ser 290 295 300 Pro Val His Asp Asp Thr
Ser Ser Asn His Thr Leu Asp Ser Asn Pro 305 310 315 320 Ala Thr Phe
Pro Leu Asn Ser Thr Leu Tyr Ala Asp Phe Ser His Asp 325 330 335 Asn
Gly Ile Ile Ser Ile Leu Phe Ala Leu Gly Leu Tyr Asn Gly Thr 340 345
350 Lys Pro Leu Ser Thr Thr Thr Val Glu Asn Ile Thr Gln Thr Asp Gly
355 360 365 Phe Ser Ser Ala Trp Thr Val Pro Phe Ala Ser Arg Leu Tyr
Val Glu 370 375 380 Met Met Gln Cys Gln Ala Glu Gln Glu Pro Leu Val
Arg Val Leu Val 385 390 395 400 Asn Asp Arg Val Val Pro Leu His Gly
Cys Pro Ile Asp Ala Leu Gly 405 410 415 Arg Cys Thr Arg Asp Ser Phe
Val Arg Gly Leu Ser Phe Ala Arg Ser 420 425 430 Gly Gly Asp Trp Ala
Glu Cys Phe Ala 435 440 5 441 PRT Aspergillus niger 5 Asn Gln Ser
Ser Cys Asp Thr Val Asp Gln Gly Tyr Gln Cys Phe Ser 1 5 10 15 Glu
Thr Ser His Leu Trp Gly Gln Tyr Ala Pro Phe Phe Ser Leu Ala 20 25
30 Asn Glu Ser Val Ile Ser Pro Glu Val Pro Ala Gly Cys Arg Val Thr
35 40 45 Phe Ala Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr
Asp Ser 50 55 60 Lys Gly Lys Lys Tyr Ser Ala Leu Ile Glu Glu Ile
Gln Gln Asn Ala 65 70 75 80 Thr Thr Phe Asp Gly Lys Tyr Ala Phe Leu
Lys Thr Tyr Asn Tyr Ser 85 90 95 Leu Gly Ala Asp Asp Leu Thr Pro
Phe Gly Glu Gln Glu Leu Val Asn 100 105 110 Ser Gly Ile Lys Phe Tyr
Gln Arg Tyr Glu Ser Leu Thr Arg Asn Ile 115 120 125 Val Pro Phe Ile
Arg Ser Ser Gly Ser Ser Arg Val Ile Ala Ser Gly 130 135 140 Lys Lys
Phe Ile Glu Gly Phe Gln Ser Thr Lys Leu Lys Asp Pro Arg 145 150 155
160 Ala Gln Pro Gly Gln Ser Ser Pro Lys Ile Asp Val Val Ile Ser Glu
165 170 175 Ala Ser Ser Ser Asn Asn Thr Leu Asp Pro Gly Thr Cys Thr
Val Phe 180 185 190 Glu Asp Ser Glu Leu Ala Asp Thr Val Glu Ala Asn
Phe Thr Ala Thr 195 200 205 Phe Val Pro Ser Ile Arg Gln Arg Leu Glu
Asn Asp Leu Ser Gly Val 210 215 220 Thr Leu Thr Asp Thr Glu Val Thr
Tyr Leu Met Asp Met Cys Ser Phe 225 230 235 240 Asp Thr Ile Ser Thr
Ser Thr Val Asp Thr Lys Leu Ser Pro Phe Cys 245 250 255 Asp Leu Phe
Thr His Asp Glu Trp Ile Asn Tyr Asp Tyr Leu Gln Ser 260 265 270 Leu
Lys Lys Tyr Tyr Gly His Gly Ala Gly Asn Pro Leu Gly Pro Thr 275 280
285 Gln Gly Val Gly Tyr Ala Asn Glu Leu Ile Ala Arg Leu Thr His Ser
290 295 300 Pro Val His Asp Asp Thr Ser Ser Asn His Thr Leu Asp Ser
Ser Pro 305 310 315 320 Ala Thr Phe Pro Leu Asn Ser Thr Leu Tyr Ala
Asp Phe Ser His Asp 325 330 335 Asn Gly Ile Ile Ser Ile Leu Phe Ala
Leu Gly Leu Tyr Asn Gly Thr 340 345 350 Lys Pro Leu Ser Thr Thr Thr
Val Glu Asn Ile Thr Gln Thr Asp Gly 355 360 365 Phe Ser Ser Ala Trp
Thr Val Pro Phe Ala Ser Arg Leu Tyr Val Glu 370 375 380 Met Met Gln
Cys Gln Ala Glu Gln Glu Pro Leu Val Arg Val Leu Val 385 390 395 400
Asn Asp Arg Val Val Pro Leu His Gly Cys Pro Val Asp Ala Leu Gly 405
410 415 Arg Cys Thr Arg Asp Ser Phe Val Arg Gly Leu Ser Phe Ala Arg
Ser 420 425 430 Gly Gly Asp Trp Ala Glu Cys Phe Ala 435 440 6 440
PRT Aspergillus fumigatus 6 Gly Ser Lys Ser Cys Asp Thr Val Asp Leu
Gly Tyr Gln Cys Ser Pro 1 5 10 15 Ala Thr Ser His Leu Trp Gly Gln
Tyr Ser Pro Phe Phe Ser Leu Glu 20 25 30 Asp Glu Leu Ser Val Ser
Ser Lys Leu Pro Lys Asp Cys Arg Ile Thr 35 40 45 Leu Val Gln Val
Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser 50 55 60 Lys Ser
Lys Lys Tyr Lys Lys Leu Val Thr Ala Ile Gln Ala Asn Ala 65 70 75 80
Thr Asp Phe Lys Gly Lys Phe Ala Phe Leu Lys Thr Tyr Asn Tyr Thr 85
90 95 Leu Gly Ala Asp Asp Leu Thr Pro Phe Gly Glu Gln Gln Leu Val
Asn 100 105 110 Ser Gly Ile Lys Phe Tyr Gln Arg Tyr Lys Ala Leu Ala
Arg Ser Val 115 120 125 Val Pro Phe Ile Arg Ala Ser Gly Ser Asp Arg
Val Ile Ala Ser Gly 130 135 140 Glu Lys Phe Ile Glu Gly Phe Gln Gln
Ala Lys Leu Ala Asp Pro Gly 145 150 155 160 Ala Thr Asn Arg Ala Ala
Pro Ala Ile Ser Val Ile Ile Pro Glu Ser 165 170 175 Glu Thr Phe Asn
Asn Thr Leu Asp His Gly Val Cys Thr Lys Phe Glu 180 185 190 Ala Ser
Gln Leu Gly Asp Glu Val Ala Ala Asn Phe Thr Ala Leu Phe 195 200 205
Ala Pro Asp Ile Arg Ala Arg Ala Glu Lys His Leu Pro Gly Val Thr
210
215 220 Leu Thr Asp Glu Asp Val Val Ser Leu Met Asp Met Cys Ser Phe
Asp 225 230 235 240 Thr Val Ala Arg Thr Ser Asp Ala Ser Gln Leu Ser
Pro Phe Cys Gln 245 250 255 Leu Phe Thr His Asn Glu Trp Lys Lys Tyr
Asn Tyr Leu Gln Ser Leu 260 265 270 Gly Lys Tyr Tyr Gly Tyr Gly Ala
Gly Asn Pro Leu Gly Pro Ala Gln 275 280 285 Gly Ile Gly Phe Thr Asn
Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro 290 295 300 Val Gln Asp His
Thr Ser Thr Asn Ser Thr Leu Val Ser Asn Pro Ala 305 310 315 320 Thr
Phe Pro Leu Asn Ala Thr Met Tyr Val Asp Phe Ser His Asp Asn 325 330
335 Ser Met Val Ser Ile Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Glu
340 345 350 Pro Leu Ser Arg Thr Ser Val Glu Ser Ala Lys Glu Leu Asp
Gly Tyr 355 360 365 Ser Ala Ser Trp Val Val Pro Phe Gly Ala Arg Ala
Tyr Phe Glu Thr 370 375 380 Met Gln Cys Lys Ser Glu Lys Glu Pro Leu
Val Arg Ala Leu Ile Asn 385 390 395 400 Asp Arg Val Val Pro Leu His
Gly Cys Asp Val Asp Lys Leu Gly Arg 405 410 415 Cys Lys Leu Asn Asp
Phe Val Lys Gly Leu Ser Trp Ala Arg Ser Gly 420 425 430 Gly Asn Trp
Gly Glu Cys Phe Ser 435 440 7 440 PRT Aspergillus fumigatus 7 Gly
Ser Lys Ser Cys Asp Thr Val Asp Leu Gly Tyr Gln Cys Ser Pro 1 5 10
15 Ala Thr Ser His Leu Trp Gly Gln Tyr Ser Pro Phe Phe Ser Leu Glu
20 25 30 Asp Glu Leu Ser Val Ser Ser Lys Leu Pro Lys Asp Cys Arg
Ile Thr 35 40 45 Leu Val Gln Val Leu Ser Arg His Gly Ala Arg Tyr
Pro Thr Ser Ser 50 55 60 Lys Ser Lys Lys Tyr Lys Lys Leu Val Thr
Ala Ile Gln Ala Asn Ala 65 70 75 80 Thr Asp Phe Lys Gly Lys Phe Ala
Phe Leu Lys Thr Tyr Asn Tyr Thr 85 90 95 Leu Gly Ala Asp Asp Leu
Thr Pro Phe Gly Glu Gln Gln Leu Val Asn 100 105 110 Ser Gly Ile Lys
Phe Tyr Gln Arg Tyr Lys Ala Leu Ala Arg Ser Val 115 120 125 Val Pro
Phe Ile Arg Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Gly 130 135 140
Glu Lys Phe Ile Glu Gly Phe Gln Gln Ala Lys Leu Ala Asp Pro Gly 145
150 155 160 Ala Thr Asn Arg Ala Ala Pro Ala Ile Ser Val Ile Ile Pro
Glu Ser 165 170 175 Glu Thr Phe Asn Asn Thr Leu Asp His Gly Val Cys
Thr Lys Phe Glu 180 185 190 Ala Ser Gln Leu Gly Asp Glu Val Ala Ala
Asn Phe Thr Ala Leu Phe 195 200 205 Ala Pro Asp Ile Arg Ala Arg Ala
Glu Lys His Leu Pro Gly Val Thr 210 215 220 Leu Thr Asp Glu Asp Val
Val Ser Leu Met Asp Met Cys Ser Phe Asp 225 230 235 240 Thr Val Ala
Arg Thr Ser Asp Ala Ser Gln Leu Ser Pro Phe Cys Gln 245 250 255 Leu
Phe Thr His Asn Glu Trp Lys Lys Tyr Asn Tyr Leu Gln Ser Leu 260 265
270 Gly Lys Tyr Tyr Gly Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln
275 280 285 Gly Ile Gly Phe Thr Asn Glu Leu Ile Ala Arg Leu Thr Arg
Ser Pro 290 295 300 Val Gln Asp His Thr Ser Thr Asn Ser Thr Leu Val
Ser Asn Pro Ala 305 310 315 320 Thr Phe Pro Leu Asn Ala Thr Met Tyr
Val Asp Phe Ser His Asp Asn 325 330 335 Ser Met Val Ser Ile Phe Phe
Ala Leu Gly Leu Tyr Asn Gly Thr Gly 340 345 350 Pro Leu Ser Arg Thr
Ser Val Glu Ser Ala Lys Glu Leu Asp Gly Tyr 355 360 365 Ser Ala Ser
Trp Val Val Pro Phe Gly Ala Arg Ala Tyr Phe Glu Thr 370 375 380 Met
Gln Cys Lys Ser Glu Lys Glu Pro Leu Val Arg Ala Leu Ile Asn 385 390
395 400 Asp Arg Val Val Pro Leu His Gly Cys Asp Val Asp Lys Leu Gly
Arg 405 410 415 Cys Lys Leu Asn Asp Phe Val Lys Gly Leu Ser Trp Ala
Arg Ser Gly 420 425 430 Gly Asn Trp Gly Glu Cys Phe Ser 435 440 8
440 PRT Aspergillus fumigatus 8 Gly Ser Lys Ser Cys Asp Thr Val Asp
Leu Gly Tyr Gln Cys Ser Pro 1 5 10 15 Ala Thr Ser His Leu Trp Gly
Gln Tyr Ser Pro Phe Phe Ser Leu Glu 20 25 30 Asp Glu Leu Ser Val
Ser Ser Lys Leu Pro Lys Asp Cys Arg Ile Thr 35 40 45 Leu Val Gln
Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser 50 55 60 Lys
Ser Lys Lys Tyr Lys Lys Leu Val Thr Ala Ile Gln Ala Asn Ala 65 70
75 80 Thr Asp Phe Lys Gly Lys Phe Ala Phe Leu Lys Thr Tyr Asn Tyr
Thr 85 90 95 Leu Gly Ala Asp Asp Leu Thr Pro Phe Gly Glu Gln Gln
Leu Val Asn 100 105 110 Ser Gly Ile Lys Phe Tyr Gln Arg Tyr Lys Ala
Leu Ala Arg Ser Val 115 120 125 Val Pro Phe Ile Arg Ala Ser Gly Ser
Asp Arg Val Ile Ala Ser Gly 130 135 140 Glu Lys Phe Ile Glu Gly Phe
Gln Gln Ala Lys Leu Ala Asp Pro Gly 145 150 155 160 Ala Thr Asn Arg
Ala Ala Pro Ala Ile Ser Val Ile Ile Pro Glu Ser 165 170 175 Glu Thr
Phe Asn Asn Thr Leu Asp His Gly Val Cys Thr Lys Phe Glu 180 185 190
Ala Ser Gln Leu Gly Asp Glu Val Ala Ala Asn Phe Thr Ala Leu Phe 195
200 205 Ala Pro Asp Ile Arg Ala Arg Ala Glu Lys His Leu Pro Gly Val
Thr 210 215 220 Leu Thr Asp Glu Asp Val Val Ser Leu Met Asp Met Cys
Ser Phe Asp 225 230 235 240 Thr Val Ala Arg Thr Ser Asp Ala Ser Gln
Leu Ser Pro Phe Cys Gln 245 250 255 Leu Phe Thr His Asn Glu Trp Lys
Lys Tyr Asn Tyr Leu Gln Ser Leu 260 265 270 Gly Lys Tyr Tyr Gly Tyr
Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln 275 280 285 Gly Ile Gly Phe
Thr Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro 290 295 300 Val Gln
Asp His Thr Ser Thr Asn Ser Thr Leu Val Ser Asn Pro Ala 305 310 315
320 Thr Phe Pro Leu Asn Ala Thr Met Tyr Val Asp Phe Ser His Asp Asn
325 330 335 Ser Met Val Ser Ile Phe Phe Ala Leu Gly Leu Tyr Asn Gly
Thr Glu 340 345 350 Pro Leu Ser Arg Thr Ser Val Glu Ser Ala Lys Glu
Leu Asp Gly Tyr 355 360 365 Ser Ala Ser Trp Val Val Pro Phe Gly Ala
Arg Ala Tyr Phe Glu Thr 370 375 380 Met Gln Cys Lys Ser Glu Lys Glu
Ser Leu Val Arg Ala Leu Ile Asn 385 390 395 400 Asp Arg Val Val Pro
Leu His Gly Cys Asp Val Asp Lys Leu Gly Arg 405 410 415 Cys Lys Leu
Asn Asp Phe Val Lys Gly Leu Ser Trp Ala Arg Ser Gly 420 425 430 Gly
Asn Trp Gly Glu Cys Phe Ser 435 440 9 440 PRT Aspergillus fumigatus
9 Gly Ser Lys Ser Cys Asp Thr Val Asp Leu Gly Tyr Gln Cys Ser Pro 1
5 10 15 Ala Thr Ser His Leu Trp Gly Gln Tyr Ser Pro Phe Phe Ser Leu
Glu 20 25 30 Asp Glu Leu Ser Val Ser Ser Lys Leu Pro Lys Asp Cys
Arg Ile Thr 35 40 45 Leu Val Gln Val Leu Ser Arg His Gly Ala Arg
Tyr Pro Thr Ser Ser 50 55 60 Lys Ser Lys Lys Tyr Lys Lys Leu Val
Thr Ala Ile Gln Ala Asn Ala 65 70 75 80 Thr Asp Phe Lys Gly Lys Phe
Ala Phe Leu Lys Thr Tyr Asn Tyr Thr 85 90 95 Leu Gly Ala Asp Asp
Leu Thr Ala Phe Gly Glu Gln Gln Leu Val Asn 100 105 110 Ser Gly Ile
Lys Phe Tyr Gln Arg Tyr Lys Ala Leu Ala Arg Ser Val 115 120 125 Val
Pro Phe Ile Arg Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Gly 130 135
140 Glu Lys Phe Ile Glu Gly Phe Gln Gln Ala Lys Leu Ala Asp Pro Gly
145 150 155 160 Ala Thr Asn Arg Ala Ala Pro Ala Ile Ser Val Ile Ile
Pro Glu Ser 165 170 175 Glu Thr Phe Asn Asn Thr Leu Asp His Gly Val
Cys Thr Lys Phe Glu 180 185 190 Ala Ser Gln Leu Gly Asp Glu Val Ala
Ala Asn Phe Thr Ala Leu Phe 195 200 205 Ala Pro Asp Ile Arg Ala Arg
Ala Lys Lys His Leu Pro Gly Val Thr 210 215 220 Leu Thr Asp Glu Asp
Val Val Ser Leu Met Asp Met Cys Ser Phe Asp 225 230 235 240 Thr Val
Ala Arg Thr Ser Asp Ala Ser Gln Leu Ser Pro Phe Cys Gln 245 250 255
Leu Phe Thr His Asn Glu Trp Lys Lys Tyr Asn Tyr Leu Gln Ser Leu 260
265 270 Gly Lys Tyr Tyr Gly Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala
Gln 275 280 285 Gly Ile Gly Phe Thr Asn Glu Leu Ile Ala Arg Leu Thr
Arg Ser Pro 290 295 300 Val Gln Asp His Thr Ser Thr Asn Ser Thr Leu
Val Ser Asn Pro Ala 305 310 315 320 Thr Phe Pro Leu Asn Ala Thr Met
Tyr Val Asp Phe Ser His Asp Asn 325 330 335 Ser Met Val Ser Ile Phe
Phe Ala Leu Gly Leu Tyr Asn Gly Thr Glu 340 345 350 Pro Leu Ser Arg
Thr Ser Val Glu Ser Ala Lys Glu Leu Asp Gly Tyr 355 360 365 Ser Ala
Ser Trp Val Val Pro Phe Gly Ala Arg Ala Tyr Phe Glu Thr 370 375 380
Met Gln Cys Lys Ser Glu Lys Glu Pro Leu Val Arg Ala Leu Ile Asn 385
390 395 400 Asp Arg Val Val Pro Leu His Gly Cys Asp Val Asp Lys Leu
Gly Arg 405 410 415 Cys Lys Leu Asn Asp Phe Val Lys Gly Leu Ser Trp
Ala Arg Ser Gly 420 425 430 Gly Asn Trp Gly Glu Cys Phe Ser 435 440
10 440 PRT Aspergillus fumigatus 10 Gly Ser Lys Ala Cys Asp Thr Val
Glu Leu Gly Tyr Gln Cys Ser Pro 1 5 10 15 Gly Thr Ser His Leu Trp
Gly Gln Tyr Ser Pro Phe Phe Ser Leu Glu 20 25 30 Asp Glu Leu Ser
Val Ser Ser Asp Leu Pro Lys Asp Cys Arg Val Thr 35 40 45 Phe Val
Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ala Ser 50 55 60
Lys Ser Lys Lys Tyr Lys Lys Leu Val Thr Ala Ile Gln Lys Asn Ala 65
70 75 80 Thr Glu Phe Lys Gly Lys Phe Ala Phe Leu Glu Thr Tyr Asn
Tyr Thr 85 90 95 Leu Gly Ala Asp Asp Leu Thr Pro Phe Gly Glu Gln
Gln Met Val Asn 100 105 110 Ser Gly Ile Lys Phe Tyr Gln Lys Tyr Lys
Ala Leu Ala Gly Ser Val 115 120 125 Val Pro Phe Ile Arg Ser Ser Gly
Ser Asp Arg Val Ile Ala Ser Gly 130 135 140 Glu Lys Phe Ile Glu Gly
Phe Gln Gln Ala Asn Val Ala Asp Pro Gly 145 150 155 160 Ala Thr Asn
Arg Ala Ala Pro Val Ile Ser Val Ile Ile Pro Glu Ser 165 170 175 Glu
Thr Tyr Asn Asn Thr Leu Asp His Ser Val Cys Thr Asn Phe Glu 180 185
190 Ala Ser Glu Leu Gly Asp Glu Val Glu Ala Asn Phe Thr Ala Leu Phe
195 200 205 Ala Pro Ala Ile Arg Ala Arg Ile Glu Lys His Leu Pro Gly
Val Gln 210 215 220 Leu Thr Asp Asp Asp Val Val Ser Leu Met Asp Met
Cys Ser Phe Asp 225 230 235 240 Thr Val Ala Arg Thr Ala Asp Ala Ser
Glu Leu Ser Pro Phe Cys Ala 245 250 255 Ile Phe Thr His Asn Glu Trp
Lys Lys Tyr Asp Tyr Leu Gln Ser Leu 260 265 270 Gly Lys Tyr Tyr Gly
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln 275 280 285 Gly Ile Gly
Phe Thr Asn Glu Leu Ile Ala Arg Leu Thr Asn Ser Pro 290 295 300 Val
Gln Asp His Thr Ser Thr Asn Ser Thr Leu Asp Ser Asp Pro Ala 305 310
315 320 Thr Phe Pro Leu Asn Ala Thr Ile Tyr Val Asp Phe Ser His Asp
Asn 325 330 335 Gly Met Ile Pro Ile Phe Phe Ala Met Gly Leu Tyr Asn
Gly Thr Glu 340 345 350 Pro Leu Ser Gln Thr Ser Glu Glu Ser Thr Lys
Glu Ser Asn Gly Tyr 355 360 365 Ser Ala Ser Trp Ala Val Pro Phe Gly
Ala Arg Ala Tyr Phe Glu Thr 370 375 380 Met Gln Cys Lys Ser Glu Lys
Glu Pro Leu Val Arg Ala Leu Ile Asn 385 390 395 400 Asp Arg Val Val
Pro Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg 405 410 415 Cys Lys
Leu Lys Asp Phe Val Lys Gly Leu Ser Trp Ala Arg Ser Gly 420 425 430
Gly Asn Ser Glu Gln Ser Phe Ser 435 440 11 439 PRT Aspergillus
nidulans 11 Gln Asn His Ser Cys Asn Thr Ala Asp Gly Gly Tyr Gln Cys
Phe Pro 1 5 10 15 Asn Val Ser His Val Trp Gly Gln Tyr Ser Pro Tyr
Phe Ser Ile Glu 20 25 30 Gln Glu Ser Ala Ile Ser Glu Asp Val Pro
His Gly Cys Glu Val Thr 35 40 45 Phe Val Gln Val Leu Ser Arg His
Gly Ala Arg Tyr Pro Thr Glu Ser 50 55 60 Lys Ser Lys Ala Tyr Ser
Gly Leu Ile Glu Ala Ile Gln Lys Asn Ala 65 70 75 80 Thr Ser Phe Trp
Gly Gln Tyr Ala Phe Leu Glu Ser Tyr Asn Tyr Thr 85 90 95 Leu Gly
Ala Asp Asp Leu Thr Ile Phe Gly Glu Asn Gln Met Val Asp 100 105 110
Ser Gly Ala Lys Phe Tyr Arg Arg Tyr Lys Asn Leu Ala Arg Lys Asn 115
120 125 Thr Pro Phe Ile Arg Ala Ser Gly Ser Asp Arg Val Val Ala Ser
Ala 130 135 140 Glu Lys Phe Ile Asn Gly Phe Arg Lys Ala Gln Leu His
Asp His Gly 145 150 155 160 Ser Gly Gln Ala Thr Pro Val Val Asn Val
Ile Ile Pro Glu Ile Asp 165 170 175 Gly Phe Asn Asn Thr Leu Asp His
Ser Thr Cys Val Ser Phe Glu Asn 180 185 190 Asp Glu Arg Ala Asp Glu
Ile Glu Ala Asn Phe Thr Ala Ile Met Gly 195 200 205 Pro Pro Ile Arg
Lys Arg Leu Glu Asn Asp Leu Pro Gly Ile Lys Leu 210 215 220 Thr Asn
Glu Asn Val Ile Tyr Leu Met Asp Met Cys Ser Phe Asp Thr 225 230 235
240 Met Ala Arg Thr Ala His Gly Thr Glu Leu Ser Pro Phe Cys Ala Ile
245 250 255 Phe Thr Glu Lys Glu Trp Leu Gln Tyr Asp Tyr Leu Gln Ser
Leu Ser 260 265 270 Lys Tyr Tyr Gly Tyr Gly Ala Gly Ser Pro Leu Gly
Pro Ala Gln Gly 275 280 285 Ile Gly Phe Thr Asn Glu Leu Ile Ala Arg
Leu Thr Gln Ser Pro Val 290 295 300 Gln Asp Asn Thr Ser Thr Asn His
Thr Leu Asp Ser Asn Pro Ala Thr 305 310 315 320 Phe Pro Leu Asp Arg
Lys Leu Tyr Ala Asp Phe Ser His Asp Asn Ser 325 330 335 Met Ile Ser
Ile Phe Phe Ala Met Gly Leu Tyr Asn Gly Thr Gln Pro 340 345 350 Leu
Ser Met Asp Ser Val Glu Ser Ile Gln Glu Met Asp Gly Tyr Ala 355 360
365 Ala Ser Trp Thr Val Pro Phe Gly Ala Arg Ala Tyr Phe Glu Leu Met
370 375 380 Gln Cys Glu Lys Lys Glu Pro Leu Val Arg Val Leu Val Asn
Asp Arg 385 390 395 400 Val Val Pro Leu His Gly Cys Ala Val Asp Lys
Phe Gly Arg Cys Thr 405 410 415 Leu Asp Asp Trp Val Glu Gly Leu Asn
Phe Ala Arg Ser Gly Gly Asn 420 425 430 Trp Lys Thr Cys Phe Thr Leu
435 12 443 PRT
Talaromyces thermophilus 12 Asp Ser His Ser Cys Asn Thr Val Glu Gly
Gly Tyr Gln Cys Arg Pro 1 5 10 15 Glu Ile Ser His Ser Trp Gly Gln
Tyr Ser Pro Phe Phe Ser Leu Ala 20 25 30 Asp Gln Ser Glu Ile Ser
Pro Asp Val Pro Gln Asn Cys Lys Ile Thr 35 40 45 Phe Val Gln Leu
Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser 50 55 60 Lys Thr
Glu Leu Tyr Ser Gln Leu Ile Ser Arg Ile Gln Lys Thr Ala 65 70 75 80
Thr Ala Tyr Lys Gly Tyr Tyr Ala Phe Leu Lys Asp Tyr Arg Tyr Gln 85
90 95 Leu Gly Ala Asn Asp Leu Thr Pro Phe Gly Glu Asn Gln Met Ile
Gln 100 105 110 Leu Gly Ile Lys Phe Tyr Asn His Tyr Lys Ser Leu Ala
Arg Asn Ala 115 120 125 Val Pro Phe Val Arg Cys Ser Gly Ser Asp Arg
Val Ile Ala Ser Gly 130 135 140 Arg Leu Phe Ile Glu Gly Phe Gln Ser
Ala Lys Val Leu Asp Pro His 145 150 155 160 Ser Asp Lys His Asp Ala
Pro Pro Thr Ile Asn Val Ile Ile Glu Glu 165 170 175 Gly Pro Ser Tyr
Asn Asn Thr Leu Asp Thr Gly Ser Cys Pro Val Phe 180 185 190 Glu Asp
Ser Ser Gly Gly His Asp Ala Gln Glu Lys Phe Ala Lys Gln 195 200 205
Phe Ala Pro Ala Ile Leu Glu Lys Ile Lys Asp His Leu Pro Gly Val 210
215 220 Asp Leu Ala Val Ser Asp Val Pro Tyr Leu Met Asp Leu Cys Pro
Phe 225 230 235 240 Glu Thr Leu Ala Arg Asn His Thr Asp Thr Leu Ser
Pro Phe Cys Ala 245 250 255 Leu Ser Thr Gln Glu Glu Trp Gln Ala Tyr
Asp Tyr Tyr Gln Ser Leu 260 265 270 Gly Lys Tyr Tyr Gly Asn Gly Gly
Gly Asn Pro Leu Gly Pro Ala Gln 275 280 285 Gly Val Gly Phe Val Asn
Glu Leu Ile Ala Arg Met Thr His Ser Pro 290 295 300 Val Gln Asp Tyr
Thr Thr Val Asn His Thr Leu Asp Ser Asn Pro Ala 305 310 315 320 Thr
Phe Pro Leu Asn Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn 325 330
335 Thr Met Thr Ser Ile Phe Ala Ala Leu Gly Leu Tyr Asn Gly Thr Ala
340 345 350 Lys Leu Ser Thr Thr Glu Ile Lys Ser Ile Glu Glu Thr Asp
Gly Tyr 355 360 365 Ser Ala Ala Trp Thr Val Pro Phe Gly Gly Arg Ala
Tyr Ile Glu Met 370 375 380 Met Gln Cys Asp Asp Ser Asp Glu Pro Val
Val Arg Val Leu Val Asn 385 390 395 400 Asp Arg Val Val Pro Leu His
Gly Cys Glu Val Asp Ser Leu Gly Arg 405 410 415 Cys Lys Arg Asp Asp
Phe Val Arg Gly Leu Ser Phe Ala Arg Gln Gly 420 425 430 Gly Asn Trp
Glu Gly Cys Tyr Ala Ala Ser Glu 435 440 13 466 PRT Myceliophthora
thermophila 13 Glu Ser Arg Pro Cys Asp Thr Pro Asp Leu Gly Phe Gln
Cys Gly Thr 1 5 10 15 Ala Ile Ser His Phe Trp Gly Gln Tyr Ser Pro
Tyr Phe Ser Val Pro 20 25 30 Ser Glu Leu Asp Ala Ser Ile Pro Asp
Asp Cys Glu Val Thr Phe Ala 35 40 45 Gln Val Leu Ser Arg His Gly
Ala Arg Ala Pro Thr Leu Lys Arg Ala 50 55 60 Ala Ser Tyr Val Asp
Leu Ile Asp Arg Ile His His Gly Ala Ile Ser 65 70 75 80 Tyr Gly Pro
Gly Tyr Glu Phe Leu Arg Thr Tyr Asp Tyr Thr Leu Gly 85 90 95 Ala
Asp Glu Leu Thr Arg Thr Gly Gln Gln Gln Met Val Asn Ser Gly 100 105
110 Ile Lys Phe Tyr Arg Arg Tyr Arg Ala Leu Ala Arg Lys Ser Ile Pro
115 120 125 Phe Val Arg Thr Ala Gly Gln Asp Arg Val Val His Ser Ala
Glu Asn 130 135 140 Phe Thr Gln Gly Phe His Ser Ala Leu Leu Ala Asp
Arg Gly Ser Thr 145 150 155 160 Val Arg Pro Thr Leu Pro Tyr Asp Met
Val Val Ile Pro Glu Thr Ala 165 170 175 Gly Ala Asn Asn Thr Leu His
Asn Asp Leu Cys Thr Ala Phe Glu Glu 180 185 190 Gly Pro Tyr Ser Thr
Ile Gly Asp Asp Ala Gln Asp Thr Tyr Leu Ser 195 200 205 Thr Phe Ala
Gly Pro Ile Thr Ala Arg Val Asn Ala Asn Leu Pro Gly 210 215 220 Ala
Asn Leu Thr Asp Ala Asp Thr Val Ala Leu Met Asp Leu Cys Pro 225 230
235 240 Phe Glu Thr Val Ala Ser Ser Ser Ser Asp Pro Ala Thr Ala Asp
Ala 245 250 255 Gly Gly Gly Asn Gly Arg Pro Leu Ser Pro Phe Cys Arg
Leu Phe Ser 260 265 270 Glu Ser Glu Trp Arg Ala Tyr Asp Tyr Leu Gln
Ser Val Gly Lys Trp 275 280 285 Tyr Gly Tyr Gly Pro Gly Asn Pro Leu
Gly Pro Thr Gln Gly Val Gly 290 295 300 Phe Val Asn Glu Leu Leu Ala
Arg Leu Ala Gly Val Pro Val Arg Asp 305 310 315 320 Gly Thr Ser Thr
Asn Arg Thr Leu Asp Gly Asp Pro Arg Thr Phe Pro 325 330 335 Leu Gly
Arg Pro Leu Tyr Ala Asp Phe Ser His Asp Asn Asp Met Met 340 345 350
Gly Val Leu Gly Ala Leu Gly Ala Tyr Asp Gly Val Pro Pro Leu Asp 355
360 365 Lys Thr Ala Arg Arg Asp Pro Glu Glu Leu Gly Gly Tyr Ala Ala
Ser 370 375 380 Trp Ala Val Pro Phe Ala Ala Arg Ile Tyr Val Glu Lys
Met Arg Cys 385 390 395 400 Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly
Glu Gly Arg Gln Glu Lys 405 410 415 Asp Glu Glu Met Val Arg Val Leu
Val Asn Asp Arg Val Met Thr Leu 420 425 430 Lys Gly Cys Gly Ala Asp
Glu Arg Gly Met Cys Thr Leu Glu Arg Phe 435 440 445 Ile Glu Ser Met
Ala Phe Ala Arg Gly Asn Gly Lys Trp Asp Leu Cys 450 455 460 Phe Ala
465 14 441 PRT Artificial Calculated consensus sequence. 14 Asn Ser
His Ser Cys Asp Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro 1 5 10 15
Glu Ile Ser His Leu Trp Gly Gln Tyr Ser Pro Tyr Phe Ser Leu Glu 20
25 30 Asp Glu Ser Ala Ile Ser Pro Asp Val Pro Asp Asp Cys Xaa Val
Thr 35 40 45 Phe Val Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro
Thr Ser Ser 50 55 60 Lys Xaa Lys Ala Tyr Ser Ala Leu Ile Glu Ala
Ile Gln Lys Asn Ala 65 70 75 80 Thr Xaa Phe Lys Gly Lys Tyr Ala Phe
Leu Lys Thr Tyr Asn Tyr Thr 85 90 95 Leu Gly Ala Asp Asp Leu Thr
Pro Phe Gly Glu Asn Gln Met Val Asn 100 105 110 Ser Gly Ile Lys Phe
Tyr Arg Arg Tyr Lys Ala Leu Ala Arg Lys Xaa 115 120 125 Val Pro Phe
Val Arg Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Ala 130 135 140 Glu
Lys Phe Ile Glu Gly Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly 145 150
155 160 Ser Xaa Pro His Gln Ala Ser Pro Val Ile Asn Val Ile Ile Pro
Glu 165 170 175 Gly Ser Gly Tyr Asn Asn Thr Leu Asp His Gly Thr Cys
Thr Ala Phe 180 185 190 Glu Asp Ser Glu Leu Gly Asp Asp Ala Glu Ala
Asn Phe Thr Ala Thr 195 200 205 Phe Ala Pro Ala Ile Arg Ala Arg Leu
Glu Ala Asp Leu Pro Gly Val 210 215 220 Thr Leu Thr Asp Glu Asp Val
Val Xaa Leu Met Asp Met Cys Pro Phe 225 230 235 240 Glu Thr Val Ala
Arg Thr Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys 245 250 255 Ala Leu
Phe Thr Glu Xaa Glu Trp Xaa Xaa Tyr Asp Tyr Leu Gln Ser 260 265 270
Leu Gly Lys Tyr Tyr Gly Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala 275
280 285 Gln Gly Val Gly Phe Xaa Asn Glu Leu Ile Ala Arg Leu Thr His
Ser 290 295 300 Pro Val Gln Asp His Thr Ser Thr Asn His Thr Leu Asp
Ser Asn Pro 305 310 315 320 Ala Thr Phe Pro Leu Asn Ala Thr Leu Tyr
Ala Asp Phe Ser His Asp 325 330 335 Asn Ser Met Ile Ser Ile Phe Phe
Ala Leu Gly Leu Tyr Asn Gly Thr 340 345 350 Ala Pro Leu Ser Thr Thr
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly 355 360 365 Tyr Ala Ala Ser
Trp Thr Val Pro Phe Gly Ala Arg Ala Tyr Val Glu 370 375 380 Met Met
Gln Cys Gln Ala Glu Lys Glu Pro Leu Val Arg Val Leu Val 385 390 395
400 Asn Asp Arg Val Val Pro Leu His Gly Cys Ala Val Asp Lys Leu Gly
405 410 415 Arg Cys Lys Leu Asp Asp Phe Val Glu Gly Leu Ser Phe Ala
Arg Ser 420 425 430 Gly Gly Asn Trp Ala Glu Cys Phe Ala 435 440 15
441 PRT Artificial Constructed consensus phytase sequence. 15 Asn
Ser His Ser Cys Asp Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro 1 5 10
15 Glu Ile Ser His Leu Trp Gly Gln Tyr Ser Pro Tyr Phe Ser Leu Glu
20 25 30 Asp Glu Ser Ala Ile Ser Pro Asp Val Pro Asp Asp Cys Arg
Val Thr 35 40 45 Phe Val Gln Val Leu Ser Arg His Gly Ala Arg Tyr
Pro Thr Ser Ser 50 55 60 Lys Ser Lys Ala Tyr Ser Ala Leu Ile Glu
Ala Ile Gln Lys Asn Ala 65 70 75 80 Thr Ala Phe Lys Gly Lys Tyr Ala
Phe Leu Lys Thr Tyr Asn Tyr Thr 85 90 95 Leu Gly Ala Asp Asp Leu
Thr Pro Phe Gly Glu Asn Gln Met Val Asn 100 105 110 Ser Gly Ile Lys
Phe Tyr Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile 115 120 125 Val Pro
Phe Ile Arg Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Ala 130 135 140
Glu Lys Phe Ile Glu Gly Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly 145
150 155 160 Ser Gln Pro His Gln Ala Ser Pro Val Ile Asp Val Ile Ile
Pro Glu 165 170 175 Gly Ser Gly Tyr Asn Asn Thr Leu Asp His Gly Thr
Cys Thr Ala Phe 180 185 190 Glu Asp Ser Glu Leu Gly Asp Asp Val Glu
Ala Asn Phe Thr Ala Leu 195 200 205 Phe Ala Pro Ala Ile Arg Ala Arg
Leu Glu Ala Asp Leu Pro Gly Val 210 215 220 Thr Leu Thr Asp Glu Asp
Val Val Tyr Leu Met Asp Met Cys Pro Phe 225 230 235 240 Glu Thr Val
Ala Arg Thr Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys 245 250 255 Ala
Leu Phe Thr His Asp Glu Trp Arg Gln Tyr Asp Tyr Leu Gln Ser 260 265
270 Leu Gly Lys Tyr Tyr Gly Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala
275 280 285 Gln Gly Val Gly Phe Ala Asn Glu Leu Ile Ala Arg Leu Thr
Arg Ser 290 295 300 Pro Val Gln Asp His Thr Ser Thr Asn His Thr Leu
Asp Ser Asn Pro 305 310 315 320 Ala Thr Phe Pro Leu Asn Ala Thr Leu
Tyr Ala Asp Phe Ser His Asp 325 330 335 Asn Ser Met Ile Ser Ile Phe
Phe Ala Leu Gly Leu Tyr Asn Gly Thr 340 345 350 Ala Pro Leu Ser Thr
Thr Ser Val Glu Ser Ile Glu Glu Thr Asp Gly 355 360 365 Tyr Ser Ala
Ser Trp Thr Val Pro Phe Gly Ala Arg Ala Tyr Val Glu 370 375 380 Met
Met Gln Cys Gln Ala Glu Lys Glu Pro Leu Val Arg Val Leu Val 385 390
395 400 Asn Asp Arg Val Val Pro Leu His Gly Cys Ala Val Asp Lys Leu
Gly 405 410 415 Arg Cys Lys Arg Asp Asp Phe Val Glu Gly Leu Ser Phe
Ala Arg Ser 420 425 430 Gly Gly Asn Trp Ala Glu Cys Phe Ala 435 440
16 1426 DNA Artificial DNA sequence of the fungal consensus gene
(fcp). 16 tatatgaatt catgggcgtg ttcgtcgtgc tactgtccat tgccaccttg
ttcggttcca 60 catccggtac cgccttgggt cctcgtggta attctcactc
ttgtgacact gttgacggtg 120 gttaccaatg tttcccagaa atttctcact
tgtggggtca atactctcca tacttctctt 180 tggaagacga atctgctatt
tctccagacg ttccagacga ctgtagagtt actttcgttc 240 aagttttgtc
tagacacggt gctagatacc caacttcttc taagtctaag gcttactctg 300
ctttgattga agctattcaa aagaacgcta ctgctttcaa gggtaagtac gctttcttga
360 agacttacaa ctacactttg ggtgctgacg acttgactcc attcggtgaa
aaccaaatgg 420 ttaactctgg tattaagttc tacagaagat acaaggcttt
ggctagaaag attgttccat 480 tcattagagc ttctggttct gacagagtta
ttgcttctgc tgaaaagttc attgaaggtt 540 tccaatctgc taagttggct
gacccaggtt ctcaaccaca ccaagcttct ccagttattg 600 acgttattat
tccagaagga tccggttaca acaacacttt ggaccacggt acttgtactg 660
ctttcgaaga ctctgaattg ggtgacgacg ttgaagctaa cttcactgct ttgttcgctc
720 cagctattag agctagattg gaagctgact tgccaggtgt tactttgact
gacgaagacg 780 ttgtttactt gatggacatg tgtccattcg aaactgttgc
tagaacttct gacgctactg 840 aattgtctcc attctgtgct ttgttcactc
acgacgaatg gagacaatac gactacttgc 900 aatctttggg taagtactac
ggttacggtg ctggtaaccc attgggtcca gctcaaggtg 960 ttggtttcgc
taacgaattg attgctagat tgactagatc tccagttcaa gaccacactt 1020
ctactaacca cactttggac tctaacccag ctactttccc attgaacgct actttgtacg
1080 ctgacttctc tcacgacaac tctatgattt ctattttctt cgctttgggt
ttgtacaacg 1140 gtactgctcc attgtctact acttctgttg aatctattga
agaaactgac ggttactctg 1200 cttcttggac tgttccattc ggtgctagag
cttacgttga aatgatgcaa tgtcaagctg 1260 aaaaggaacc attggttaga
gttttggtta acgacagagt tgttccattg cacggttgtg 1320 ctgttgacaa
gttgggtaga tgtaagagag acgacttcgt tgaaggtttg tctttcgcta 1380
gatctggtgg taactgggct gaatgtttcg cttaagaatt catata 1426 17 467 PRT
Artificial Constructed consensus phytase sequence with signal
peptide of phytase from A. terreus cbs fused to N-terminus. 17 Met
Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser 1 5 10
15 Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
20 25 30 Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His
Leu Trp 35 40 45 Gly Gln Tyr Ser Pro Tyr Phe Ser Leu Glu Asp Glu
Ser Ala Ile Ser 50 55 60 Pro Asp Val Pro Asp Asp Cys Arg Val Thr
Phe Val Gln Val Leu Ser 65 70 75 80 Arg His Gly Ala Arg Tyr Pro Thr
Ser Ser Lys Ser Lys Ala Tyr Ser 85 90 95 Ala Leu Ile Glu Ala Ile
Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys 100 105 110 Tyr Ala Phe Leu
Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu 115 120 125 Thr Pro
Phe Gly Glu Asn Gln Met Val Asn Ser Gly Ile Lys Phe Tyr 130 135 140
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala 145
150 155 160 Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile
Glu Gly 165 170 175 Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ser Gln
Pro His Gln Ala 180 185 190 Ser Pro Val Ile Asp Val Ile Ile Pro Glu
Gly Ser Gly Tyr Asn Asn 195 200 205 Thr Leu Asp His Gly Thr Cys Thr
Ala Phe Glu Asp Ser Glu Leu Gly 210 215 220 Asp Asp Val Glu Ala Asn
Phe Thr Ala Leu Phe Ala Pro Ala Ile Arg 225 230 235 240 Ala Arg Leu
Glu Ala Asp Leu Pro Gly Val Thr Leu Thr Asp Glu Asp 245 250 255 Val
Val Tyr Leu Met Asp Met Cys Pro Phe Glu Thr Val Ala Arg Thr 260 265
270 Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys Ala Leu Phe Thr His Asp
275 280 285 Glu Trp Arg Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr
Tyr Gly 290 295 300 Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly
Val Gly Phe Ala 305 310 315 320 Asn Glu Leu Ile Ala Arg Leu Thr Arg
Ser Pro Val Gln Asp His Thr 325 330 335 Ser Thr Asn His Thr Leu Asp
Ser Asn Pro Ala Thr Phe Pro Leu Asn 340 345 350 Ala Thr Leu Tyr Ala
Asp Phe Ser His Asp Asn Ser Met Ile Ser Ile 355 360 365 Phe Phe Ala
Leu Gly Leu Tyr
Asn Gly Thr Ala Pro Leu Ser Thr Thr 370 375 380 Ser Val Glu Ser Ile
Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr 385 390 395 400 Val Pro
Phe Gly Ala Arg Ala Tyr Val Glu Met Met Gln Cys Gln Ala 405 410 415
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro 420
425 430 Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys Arg Asp
Asp 435 440 445 Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn
Trp Ala Glu 450 455 460 Cys Phe Ala 465 18 26 DNA Artificial PCR
primer. 18 tatatgaatt catgggcgtg ttcgtc 26 19 22 DNA Artificial PCR
primer. 19 tgaaaagttc attgaaggtt tc 22 20 22 DNA Artificial PCR
primer. 20 tcttcgaaag cagtacaagt ac 22 21 22 DNA Artificial PCR
primer. 21 tatatgaatt cttaagcgaa ac 22 22 33 DNA Artificial PCR
primer. 22 tatatcatga gcgtgttcgt cgtgctactg ttc 33 23 33 DNA
Artificial PCR primer. 23 acccgactta caaagcgaat tctatagata tat 33
24 32 DNA Artificial Primer for site-directed mutagenesis. 24
cacttgtggg gtttgtacag tccatacttc tc 32 25 32 DNA Artificial Primer
for site-directed mutagenesis. 25 gagaagtatg gactgtacaa accccacaag
tg 32 26 32 DNA Artificial Primer for site-directed mutagenesis. 26
cacttgtggg gtacctactc tccatacttc tc 32 27 32 DNA Artificial Primer
for site-directed mutagenesis. 27 gagaagtatg gagagtaggt accccacaag
tg 32 28 32 DNA Artificial Primer for site-directed mutagenesis. 28
cacttgtggg gtggttactc tccatacttc tc 32 29 32 DNA Artificial Primer
for site-directed mutagenesis. 29 gagaagtatg gagagtaacc accccacaag
tg 32 30 32 DNA Artificial Primer for site-directed mutagenesis. 30
cacttgtggg gtaccaactc tccatacttc tc 32 31 32 DNA Artificial Primer
for site-directed mutagenesis. 31 gagaagtatg gagagttggt accccacaag
tg 32 32 32 DNA Artificial Primer for site-directed mutagenesis. 32
cacttgtggg gtctcaactc tccatacttc tc 32 33 32 DNA Artificial Primer
for site-directed mutagenesis. 33 gagaagtatg gagagttgag accccacaag
tg 32
* * * * *