Note: Descriptions are shown in the official language in which they were submitted.
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HAI~OPEROXIDASES WITH ALTERED pH PROFILES
FIELD OF THE INVENTION
The present invention relates to haloperoxidase variants
with an altered pH optimum compared to the wild type.
BACKGROUND OF THE INVENTION
Haloperoxidases form a class of enzymes which are able to
oxidize halides (X - C1-, Br-, or I-) in the presence of
hydrogen peroxide to the corresponding hypohalous acid (HOX)
according to:
H202 + X- + H+ -> H20 + HOX
If a convenient nucleophilic acceptor is present, a
reaction will occur with HOX whereby a diversity of halogenated
reaction products may be formed.
A chloride peroxidase (EC 1.11.1.10) is an enzyme
capable of oxidizing chloride, bromide and iodide ions with the
consumption of H202 .
A bromide peroxidase is an enzyme capable of
oxidizing bromide and iodide ions with the consumption of H202.
A iodide peroxidase (EC 1.12.1.8) is an enzyme
capable of oxidizing iodide ions with the consumption of H202.
Vanadium haloperoxidases are different from other
haloperoxidases in that the prosthetic group in theses enzymes
have structural features similar to vanadate (vanadium V),
whereas the other haloperoxidases are hemeperoxidases.
Haloperoxidases have been isolated from various
organisms: mammals, marine animals, plants, algae, a lichen,
fungi and bacteria (for reference see Biochimica et Biophysica
Acta 1161, 1993, pp. 249-256). It is generally accepted that
haloperoxidases are the enzymes responsible for the formation
of halogenated compounds in nature, although other enzymes may
be involved.
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The amino acid sequence (SEQ No. 1) for the vanadium-
containing chloroperoxidase from the fungus Curvularia
inaequalis has been published (see SWISS-PROT:P49053).
The amino acid sequence (SEQ No. 2) for the vanadium
containing chloroperoxidase from the fungus Curvularia
verruculosa has been published (see WO 97/04102).
The X-ray structure of the vanadium-containing
chloroperoxidase from the fungus Curvularia inaequalis has been
published (Proc. Natl. Acad. Sci. U S A, 93(1), 1996, 392-396;
and pdblvnc . ent ) .
Haloperoxidases are of current interest because of
their broad range of potential industrial uses. For example,
haloperoxidases have been proposed for use as an anti-microbial
agent.
BRIEF DISCLOSURE OF THE INVENTION
The present invention relates to vanadium-containing
haloperoxidase variants with an altered pH optimum compared to
the parent haloperoxidase, so in particular the present
invention deals with:
A variant of a parent vanadium-containing haloperoxidase, which
variant has haloperoxidase activity and an altered pH optimum
and comprises a mutation in a position corresponding to at
least one of the following positions:
R4 90A, L, I, Q, M, E, D;
A399G;
F397N, Y, E, Q
P395A, S;
R360A, L, I, Q, M, E, D;
K353Q,M;
S402A, T, V, S;
D292L;
A501S~
W350F, Y;
V495A,T,V,S~
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K394 A, L, I, Q, M, E, D:
wherein the parent haloperoxidase has the amino acid sequence
given in SEQ ID No. 1 or the parent haloperoxidase has an amino
acid sequence which is at least 80% homologous to SEQ ID No. 1.
DETAILED DISCLOSURE OF TEE INVENTION
Homologous vanadium-containing haloperoxidases
A number of vanadium-containing haloperoxidases produced by
different fungi are homologous on the amino acid level.
An alignment of the Curvularia inaequalis and the Curvularia
verruculosa haloperoxidases was performed. The alignment uses
the haloperoxidase amino acid sequence obtained from the 3D
structure file of C. inaequalis (Brookhaven databank file
pdblvnc.ent).
When using the homology percent obtained from UWGCG program
using the GAP program with the default parameters (penalties:
gap weight=3.0, length weight=0.1: WISCONSIN PACKAGE Version
8.1-UNIX, August 1995, Genetics Computer Group, 575 Science
Drive, Madison, Wisconsin, USA 53711) the following homology
was found:
Curvularia inaequalis vanadium-containing haloperoxidase
comprising the amino acid sequence shown in SEQ ID No. 1: 100%:
Curvularia verruculosa vanadium-containing haloperoxidase
comprising the amino acid sequence shown in SEQ ID No. 2: 96%.
In the present context, "derived from" is intended not only
to indicate a vanadium-containing haloperoxidase produced or
producible by a strain of the organism in question, but also a
vanadium-containing haloperoxidase encoded by a DNA sequence
isolated from such strain and produced in a host organism
containing said DNA sequence. Finally, the term is intended to
indicate a vanadium-containing haloperoxidase which is encoded
by a DNA sequence of synthetic and/or cDNA origin and which has
the identifying characteristics of the vanadium-containing
haloperoxidase in question.
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Variants with altered pH optimum
The desired pH optimum of a vanadium-containing
haloperoxidase depends on which application is of interest,
e.g., if the vanadium-containing haloperoxidase is to be used
for denim bleaching the preferred pH optimum will be around pH
5-8, whereas if the vanadium-containing haloperoxidase is to be
used for washing purposes the preferred pH optimum will be
around pH 8-10.
It is possible to alter the pH optimum of a parent vanadium
containing haloperoxidase wherein said variant is the result of
a mutation, i.e. one or more amino acid residues have been
deleted from, replaced or added to the parent vanadium
containing haloperoxidase. By introducing charge changes in the
neighbourhood of the active site residues, the pKa of the
residue of interest can be changed in order to accomodate an
altered activity profile of the haloperoxidase in question.
It is a common belief that by introducing more negative
charged residues close to the His (the active site of the
haloperoxidase), its pKa is elevated and it will thus be able
to act in catalysis at a higher pH than previously. The active
site His will, by introducing more positive charged residues
close to His, alter its pKa to a lower pKa than previously and
thus be able to act in catalysis at a lower pH than previously.
The increase in pKa can also be obtained by decreasing the
solvent accessibility of the active site (His). The decrease in
pKa can also be obtained by increasing the solvent
accessibility of the active site (His).
But according to the present invention it is found that of
most importance is that the residues are within 10 ~ around His
496 and His 409. These residues are: 46-48, 193, 257, 259-265,
267-269, 285-294, 297-304, 307, 242, 245-346, 349-350, 353,
358-363, 365, 378, 380-384, 393-412, 441, 443, 482-502, 507,
551-557. Changes within this region are found to alter the pH
dependent activity or change the pH-optimum of the enzyme.
Residues can in this way be mutated in e.g. the Curvularia
inaequa~is haloperoxidase. Homologous structures which are
assumed to have similar structure can be modelbuild (see
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Example 1) and regions of interest found in the same way.
Preferred positions for mutations are the following:
R490A, L, I, Q, M, E, D:
5 A399G:
F397N,Y,E,Q;
P395A,S;
R360A, L, I, Q, M, E, D;
K353Q, M;
S402A, T, V, S
D292L,E;
A501S;
W350F, Y;
V495A, T, V, S:
K394 A, L, I, Q, M, E, D;
wherein the parent haloperoxidase has the amino acid sequence
given in SEQ ID No. 1, or the homologous positions in a parent
haloperoxidase which has an amino acid sequence which is at
least 80$ homologous to SEQ ID No. 1, or the homologous
positions in a parent haloperoxidase which has an amino acid
sequence which is at least 85~ homologous to SEQ ID No. 1, or
the homologous positions in a parent haloperoxidase which has
an amino acid sequence which is at least 90~ homologous to SEQ
ID No. 1, or the homologous positions in a parent
haloperoxidase which has an amino acid sequence which is at
least 95~ homologous to SEQ ID No. 1, or the homologous
positions in a parent haloperoxidase which has an amino acid
sequence which is at least 96~ homologous to SEQ ID No. 1, or
the homologous positions in a parent haloperoxidase which has
an amino acid sequence which is at least 97~ homologous to SEQ
ID No. 1, or the homologous positions in a parent
haloperoxidase which has an amino acid sequence which is at
least 98~ homologous to SEQ ID No. 1, or the homologous
positions in a parent haloperoxidase which has an amino acid
sequence which is at least 99~ homologous to SEQ ID No. 1.
In particular the following mutations are preferred:
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R487A, L, I, Q, M, E, D:
A396G:
F394N, Y, E, Q;
P392A, S;
R357A, L, I, Q, M, E, D:
K350Q,M:
S399A,T,V,S:
D289L,E;
A498S;
W347F, Y;
V492A,T,V,S;
K391A, L, I, Q, M, E, D:
wherein the parent haloperoxidase has the amino acid sequence
given in SEQ ID No. 2.
In a preferred embodiment two or more amino acid residues
may be substituted as follows:
R490A + D292L:
R490A + D292E;
R490L + D292L:
R490L + D292E;
R490I + D292L;
R490I + D292E:
R490Q + D292L;
R490Q + D292E:
R490M + D292L;
R490M + D292E;
R490E + D292L:
R490E + D292E;
R490D + D292L:
R490D + D292E;
wherein the parent haloperoxidase has the amino acid sequence
given in SEQ ID No. 1, or the homologous positions in a parent
haloperoxidase which has an amino acid sequence which is at
least 80$ homologous to SEQ ID No. 1, or the homologous
positions in a parent haloperoxidase which has an amino acid
sequence which is at least 85~ homologous to SEQ ID No. 1, or
the homologous positions in a parent haloperoxidase which has
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an amino acid sequence which is at least 90~ homologous to SEQ
ID No. 1, or the homologous positions in a parent
haloperoxidase which has an amino acid sequence which is at
least 95~ homologous to SEQ ID No. 1, or the homologous
positions in a parent haloperoxidase which has an amino acid
sequence which is at least 96~ homologous to SEQ ID No. 1, or
the homologous positions in a parent haloperoxidase which has
an amino acid sequence which is at least 97~ homologous to SEQ
ID No. 1, or the homologous positions in a parent
haloperoxidase which has an amino acid sequence which is at
least 98~ homologous to SEQ ID No. 1, or the homologous
positions in a parent haloperoxidase which has an amino acid
sequence which is at least 99~ homologous to SEQ ID No. 1.
In a preferred embodiment two or more amino acid residues
i5 may be substituted as follows:
R487A + D289L;
R987A + D289E;
R487L + D289L;
R487L + D289E;
R487I + D289L:
R487I + D289E;
R487Q + D289L;
R487Q + D289E;
R487M + D289L;
R487M + D289E;
R487E + D289L;
R487E + D289E:
R487D + D289L;
R487D + D289E;
wherein the parent haloperoxidase has the amino acid sequence
given in SEQ ID No. 2.
Methods of preparing vanadium-containing haloperoxidase
variants
Several methods for introducing mutations into genes are
known in the art. After a brief discussion of the cloning of
haloperoxidase-encoding DNA sequences, methods for generating
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mutations at specific sites within the haloperoxidase-encoding
sequence will be discussed.
Cloning a DNA sequence encoding a vanadium-containing
haloperoxidase
The DNA sequence encoding a parent vanadium-containing
haloperoxidase may be isolated from any cell or microorganism
producing the haloperoxidase in question, using various methods
well known in the art. First, a genomic DNA and/or cDNA library
should be constructed using chromosomal DNA or messenger RNA
from the organism that produces the haloperoxidase to be
studied. Then, if the amino acid sequence of the haloperoxidase
is known, homologous, labelled oligonucleotide probes may be
synthesized and used to identify haloperoxidase-encoding clones
from a genomic library prepared from the organism in question.
Alternatively, a labelled oligonucleotide probe containing
sequences homologous to a known haloperoxidase gene could be
used as a probe to identify haloperoxidase-encoding clones,
using hybridization and washing conditions of lower stringency.
A method for identifying haloperoxidase-encoding clones
involves inserting cDNA into an expression vector, such as a
plasmid, transforming haloperoxidase-negative fungi with the
resulting cDNA library, and then plating the transformed fungi
onto agar containing a substrate for the haloperoxidase,
thereby allowing clones expressing the haloperoxidase to be
identified.
Alternatively, the DNA sequence encoding the enzyme may be
prepared synthetically by established standard methods, e.g.
the phosphoroamidite method. In the phosphoroamidite method,
oligonucleotides are synthesized, e.g. in an automatic DNA
synthesizer, purified, annealed, ligated and cloned in appro-
priate vectors.
Finally, the DNA sequence may be of mixed genomic and syn
thetic origin, mixed synthetic and cDNA origin or mixed genomic
and cDNA origin, prepared by ligating fragments of synthetic,
genomic or cDNA origin (as appropriate, the fragments
corresponding to various parts of the entire DNA sequence), in
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accordance with standard techniques. The DNA sequence may also
be prepared by polymerase chain reaction (PCR) using specific
primers.
Site-directed mutagenesis
Once a haloperoxidase-encoding DNA sequence has been
isolated, and desirable sites for mutation identified, muta-
tions may be introduced using synthetic oligonucleotides. These
oligonucleotides contain nucleotide sequences flanking the
desired mutation sites: mutant nucleotides are inserted during
oligonucleotide synthesis. In a specific method, a single-
stranded gap of DNA, bridging the haloperoxidase-encoding
sequence, is created in a vector carrying the haloperoxidase
gene. Then the synthetic nucleotide, bearing the desired
mutation, is annealed to a homologous portion of the single-
stranded DNA. The remaining gap is then filled in with T7 DNA
polymerase and the construct is ligated using T4 ligase
(Morinaga method - see Biotechnology, 2, 1984, pp. 626-639). US
4,760,025 discloses the introduction of oligonucleotides
encoding multiple mutations by performing minor alterations of
the cassette. However, an even greater variety of mutations can
be introduced at any one time by the Morinaga method, because a
multitude of oligonucleotides, of various lengths, can be
introduced.
Another method of introducing mutations into haloperoxidase-
encoding DNA sequences is the 3-step generation of a PCR
fragment containing the desired mutation introduced by using a
chemically synthesized DNA strand as one of the primers in the
PCR reactions. From the PCR-generated fragment, a DNA fragment
carrying the mutation may be isolated by cleavage with restric
tion endonucleases and reinserted into an expression plasmid.
Random mutagenesis
The random mutagenesis of a DNA sequence encoding a parent
haloperoxidase may conveniently be performed by use of any
method known in the art.
For instance, the random mutagenesis may be performed by use
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of a suitable physical or chemical mutagenizing agent, by use
of a suitable oligonucleotide, or by subjecting the DNA
sequence to PCR generated mutagenesis. Furthermore, the random
mutagenesis may be performed by use of any combination of these
5 mutagenizing agents.
The mutagenizing agent may, e.g., be one which induces tran-
sitions, transversions, inversions, scrambling, deletions,
and/or insertions.
Examples of a physical or chemical mutagenizing agent
10 suitable for the present purpose include ultraviolet (UV) ir
radiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane
sulphonate (EMS), sodium bisulphite, formic acid, and
nucleotide analogues.
When such agents are used, the mutagenesis is typically per
formed by incubating the DNA sequence encoding the parent
enzyme to be mutagenized in the presence of the mutagenizing
agent of choice under suitable conditions for the mutagenesis
to take place, and selecting for mutated DNA having the desired
properties .
When the mutagenesis is performed by the use of an oligo-
nucleotide, the oligonucleotide may be doped or spiked with the
three non-parent nucleotides during the synthesis of the
oligonucleotide at the positions which are to be changed. The
doping or spiking may be done so that codons for unwanted amino
acids are avoided. The doped or spiked oligonucleotide can be
incorporated into the DNA encoding the haloperoxidase enzyme by
any published technique, using e.g. PCR, LCR or any DNA
polymerase and lipase.
When PCR-generated mutagenesis is used, either a chemically
treated or non-treated gene encoding a parent haloperoxidase
enzyme is subjected to PCR under conditions that increase the
misincorporation of nucleotides (Deshler 1992; Leung et al.,
Technique, Vol.l, 1989, pp. 11-15).
A mutator strain of E. coli (Fowler et al., Molec. Gen.
Genet., 133, 1974, pp. 179-191), S. ce.reviseae or any other
microbial organism may be used for the random mutagenesis of
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the DNA encoding the haloperoxidase enzyme by e.g. transforming
a plasmid containing the parent enzyme into the mutator strain,
growing the mutator strain with the plasmid and isolating the
mutated plasmid from the mutator strain. The mutated plasmid
may subsequently be transformed into the expression organism.
The DNA sequence to be mutagenized may conveniently be
present in a genomic or cDNA library prepared from an organism
expressing the parent haloperoxidase enzyme. Alternatively, the
DNA sequence may be present on a suitable vector such as a
plasmid or a bacteriophage, which as such may be incubated with
or otherwise exposed to the mutagenizing agent. The DNA to be
mutagenized may also be present in a host cell either by being
integrated in the genome of said cell or by being present on a
vector harboured in the cell. Finally, the DNA to be mutage-
nized may be in isolated form. It will be understood that the
DNA sequence to be subjected to random mutagenesis is pre-
ferably a cDNA or a genomic DNA sequence.
In some cases it may be convenient to amplify the mutated
DNA sequence prior to the expression step or the screening step
being performed. Such amplification may be performed in
accordance with methods known in the art, the presently
preferred method being PCR-generated amplification using
oligonucleotide primers prepared on the basis of the DNA or
amino acid sequence of the parent enzyme.
Subsequent to the incubation with or exposure to the mutage-
nizing agent, the mutated DNA is expressed by culturing a
suitable host cell carrying the DNA sequence under conditions
allowing expression to take place. The host cell used for this
purpose may be one which has been transformed with the mutated
DNA sequence, optionally present on a vector, or one which was
carried the DNA sequence encoding the parent enzyme during the
mutagenesis treatment. Examples of suitable host cells are
fungal hosts such as Aspergillus niger or Aspergillus oryzae.
The mutated DNA sequence may further comprise a DNA sequence
encoding functions permitting expression of the mutated DNA
sequence.
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Localized random mutagenesis
The random mutagenesis may advantageously be localized to a
part of the parent haloperoxidase in question. This may, e.g.,
be advantageous when certain regions of the enzyme have been
identified to be of particular importance for a given property
of the enzyme, and when modified are expected to result in a
variant having improved properties. Such regions may normally
be identified when the tertiary structure of the parent enzyme
has been elucidated and related to the function of the enzyme.
The localized random mutagenesis is conveniently performed
by use of PCR-generated mutagenesis techniques as described
above or any other suitable technique known in the art.
Alternatively, the DNA sequence encoding the part of the DNA
sequence to be modified may be isolated, e.g. by being inserted
into a suitable vector, and said part may subsequently be sub
jected to mutagenesis by use of any of the mutagenesis methods
discussed above.
With respect to the screening step in the above-mentioned
method of the invention, this may conveniently be performed by
use of as filter assay based on the following principle:
A microorganism capable of expressing the mutated
haloperoxidase enzyme of interest is incubated on a suitable
medium and under suitably conditions for the enzyme to be
secreted, the medium being provided with a double filter
comprising a first protein-binding filter and on top of that a
second filter exhibiting a low protein binding capability. The
microorganism is located on the second filter. Subsequent to
the incubation, the first filter comprising enzymes secreted
from the microorganisms is separated from the second filter
comprising the microorganisms. The first filter is subjected to
screening for the desired enzymatic activity and the
corresponding microbial colonies present on the second filter
are identified.
The filter used for binding the enzymatic activity may be
any protein binding filter e.g. nylon or nitrocellulose. The
top filter carrying the colonies of the expression organism may
be any filter that has no or low affinity for binding proteins
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e.g. cellulose acetate or Durapore'~'. The filter may be
pretreated with any of the conditions to be used for screening
or may be treated during the detection of enzymatic activity.
The enzymatic activity may be detected by a dye,
fluorescence, precipitation, pH indicator, IR-absorbance or any
other known technique for detection of enzymatic activity.
The detecting compound may be immobilized by any
immobilizing agent, e.g., agarose, agar, gelatine,
polyacrylamide, starch, filter paper, cloth: or any combination
of immobilizing agents.
_Expression of haloperoxidase variants
According to the invention, a DNA sequence encoding the
variant produced by methods described above, or by any alterna
tive methods known in the art, can be expressed, in enzyme
form, using an expression vector which typically includes
control sequences encoding a promoter, operator, ribosome
binding site, translation initiation signal, and, optionally, a
repressor gene or various activator genes.
The recombinant expression vector carrying the DNA sequence
encoding a haloperoxidase variant of the invention may be any
vector which may conveniently be subjected to recombinant DNA
procedures, and the choice of vector will often depend on the
host cell into which it is to be introduced. Thus, the vector
may be an autonomously replicating vector, i.e. a vector which
exists as an extrachromosomal entity, the replication of which
is independent of chromosomal replication, e.g. a plasmid, a
bacteriophage or an extrachromosomal element, minichromosome or
an artificial chromosome. Alternatively, the vector may be one
which, when introduced into a host cell, is integrated into the
host cell genome and replicated together with the chromosomes)
into which it has been integrated.
In the vector, the DNA sequence should be operably connected
to a suitable promoter sequence. The promoter may be any DNA
sequence which shows transcriptional activity in the host cell
of choice and may be derived from genes encoding proteins
either homologous or heterologous to the host cell. Examples of
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suitable promoters for directing the transcription of the DNA
sequence encoding a haloperoxidase variant of the invention,
especially in a fungal host, are those derived from the gene
encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic
proteinase, A. niger neutral a-amylase, A. niger acid stable a-
amyiase, A. niger glucoamylase, Rhizomucor miehei lipase, A.
oryzae alkaline protease, A. oryzae triose phosphate isomerase
or A. nidulans acetamidase.
The expression vector of the invention may also comprise a
suitable transcription terminator and, in eukaryotes, poly
adenylation sequences operably connected to the DNA sequence
encoding the haloperoxidase variant of the invention. Termina
tion and polyadenylation sequences may suitably be derived from
the same sources as the promoter.
The vector may further comprise a DNA sequence enabling the
vector to replicate in the host cell in question. Examples of
such sequences are the origins of replication of plasmids
pUCl9, pACYC177, pUB110, pE194, pAMBl and pIJ702.
The vector may also comprise a selectable marker, e.g. a
gene, the product of which complements a defect in the host
cell, such as one which confers antibiotic resistance such as
ampicillin, kanamycin, chloramphenicol or tetracyclin
resistance. Furthermore, the vector may comprise Aspergillus
selection markers such as amdS, argB, niaD and sC, a marker
giving rise to hygromycin resistance, or the selection may be
accomplished by co-transformation, e.g. as described in WO
91/17243.
The procedures used to ligate the DNA construct of the
invention encoding a haloperoxidase variant, the promoter,
terminator and other elements, respectively, and to insert them
into suitable vectors containing the information necessary for
replication, are well known to persons skilled in the art (cf.,
for instance, Sambrook et al. (1989)).
The cell of the invention, either comprising a DNA construct
or an expression vector of the invention as defined above, is
advantageously used as a host cell in the recombinant produc
tion of a haloperoxidase variant of the invention. The cell may
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be transformed with the DNA construct of the invention encoding
the variant, conveniently by integrating the DNA construct (in
one or more copies) in the host chromosome. This integration is
generally considered to be an advantage as the DNA sequence is
5 more likely to be stably maintained in the cell. Integration of
the DNA constructs into the host chromosome may be performed
according to conventional methods, e.g. by homologous or
heterologous recombination. Alternatively, the cell may be
transformed with an expression vector as described above in
l0 connection with the different types of host cells.
The cell of the invention may be a cell of a higher organism
such as a mammal or an insect, but is preferably a microbial
cell, e.g. a fungal cell.
The filamentous fungus may advantageously belong to a
i5 species of Aspergillus, e.g. Aspergillus oryzae or Aspergillus
niger. Fungal cells may be transformed by a process involving
protoplast formation and transformation of the protoplasts fol
lowed by regeneration of the cell wall in a manner known per
se. A suitable procedure for transformation of Aspergillus host
cells is described in EP 238 023.
In a yet further aspect, the present invention relates to a
method of producing a haloperoxidase variant of the invention,
which method comprises cultivating a host cell as described
above under conditions conducive to the production of the
variant and recovering the variant from the cells and/or cul-
ture medium.
The medium used to cultivate the cells may be any conven-
tional medium suitable for growing the host cell in question
and obtaining expression of the haloperoxidase variant of the
invention. Suitable media are available from commercial
suppliers or may be prepared according to published recipes
(e. g. as described in catalogues of the American Type Culture
Collection).
The haloperoxidase variant secreted from the host cells may
conveniently be recovered from the culture medium by well-known
procedures, including separating the cells from the medium by
centrifugation or filtration, and precipitating proteinaceous
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components of the medium by means of a salt such as ammonium
sulphate, followed by the use of chromatographic procedures
such as ion exchange chromatography, affinity chromatography,
or the like.
Industrial Applications
The haloperoxidase of the invention may be incorporated
into a detergent or cleaning composition comprising other
enzyme types useful in detergent or cleaning compositions,
preferably at least one further enzyme selected from the group
consisting of proteases, amylases, cutinases, peroxidases,
oxidases, laccases, cellulases, xylanases, and lipases. In
particular, the haloperoxidase of the invention may be used
for bleaching and for sanitation purposes.
When used for preservation of food, beverages, cosmetics
such as lotions, creams, gels, ointments, soaps, shampoos,
conditioners, antiperspirants, deodorants, mouth wash, contact
lens products, enzyme formulations, or food ingredients, the
haloperoxidase of the invention may be incorporated into the
e.g. unpreserved food, beverages, cosmetics, contact lens
products, food ingredients or anti-inflammatory product in an
amount effective for killing or inhibiting growing of
microbial cells.
Thus, the haloperoxidase used in the method of the inven
tion may by useful as a disinfectant, e.g., in the treatment
of acne, infections in the eye or the mouth, skin infections;
in antiperspirants or deodorants: in foot bath salts: far
cleaning end disinfection of contact lenses, hard surfaces,
teeth (oral care), wounds, bruises and the like.
In general it is contemplated that the haloperoxidase of
the present invention is useful for cleaning, disinfecting or
inhibiting microbial growth on any hard surface. Examples of
surfaces, which may advantageously be contacted with the
composition of the invention are surfaces of process equipment
used e.g. dairies, chemical or pharmaceutical process plants,
water sanitation systems, paper pulp processing plants, water
treatment plants, and cooling towers. The haloperoxidase of
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WO 99/47651 PCT/DK99/00133
17
the invention should be used in an amount, which is effective
for cleaning, disinfecting or inhibiting microbial growth on
the surface in question.
Further, it is contemplated that the haloperoxidase of
the invention can advantageously be used in a cleaning-in
place (C.I.P.) system for cleaning of process equipment of any
kind.
The haloperoxidase of the invention may additionally be
used for cleaning surfaces and cooking utensils in food
processing plants and in any area in which food is prepared or
served such as hospitals, nursing homes, restaurants, especial-
ly fast food restaurants, delicatessens and the like. It may
also be used as an antimicrobial in food products and would be
especially useful as a surface antimicrobial in cheeses, fruits
and vegetables and food on salad bars.
It may also be used as a preservation agent or a
disinfection agent in water based paints:
Conservation/preservation of paints
Conservation of paint products in cans has in the art
been accomplished by adding non-enzymatic organic biocides to
the paints. In the context of the invention paint is construed
as a substance comprising a solid colouring matter dissolved
or dispersed in a liquid vehicle such as water, organic
solvent and/or oils, which when spread over a surface, dries
to leave a thin coloured, decorative and/or protective
coating. Typically isothiazoliones, such as 5-chlor-2-methyl-
4-thia-zoli-3-on, has been added to the paint as biocides at
dosages in the range of about 0.05-0.5~ to inhibit/prevent
microbial growth in the paint. The method of the invention can
however suitably be applied in this field, thereby solving the
problem of the ever present environmental bio-hazards of using
toxic organic biocides by replacing these toxic biocides with
environmentally compatible enzymes. Thus the invention
provides a method for conservation of a paint comprising
contacting said paint with a haloperoxidase variant according
to the invention. Further the invention provides a paint
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WO 99/47651 PCT1DK99/00133
18
composition comprising a haloperoxidase variant according to
the invention.
The paint is preferably a water based paint, i.e. the
solids of the paint is dispersed in an aqueous solution. The
paint may contain 0-20 ~ organic solvent, preferable 0-10~,
e.g. 0-5~.
The enzyme may be added to the paint in an amount of
0.0001-100 mg active enzyme protein per liter paint,
preferable 0.001-10 mg/liter, e.g. 0.01-1 mg/liter.
Hydrogen Peroxide Sources
According to the invention the hydrogen peroxide
needed for the reaction with the haloperoxidase may be obtained
in many different ways: It may be hydrogen peroxide or a
hydrogen peroxide precursor, such as, e.g., percarbonate or
perborate, or a peroxycarboxylic acid or a salt thereof, or it
may be a hydrogen peroxide generating enzyme system, such as,
e.g., an oxidase and its substrate. Useful oxidases may be,
e.g., a glucose oxidase, a glycerol oxidase or an amino acid
oxidase. An example of an amino acid oxidase is given in WO
94/25574.
It may be advantageous to use enzymatically
generated hydrogen peroxide, since this source results in a
relatively low concentration of hydrogen peroxide under the
biologically relevant conditions. Low concentrations of
hydrogen peroxide result in an increase in the rate of
haloperoxidase-catalysed reaction.
According to the invention the hydrogen peroxide
source needed for the reaction with the haloperoxidase may be
added in a concentration corresponding to a hydrogen peroxide
concentration in the range of from 0.01-1000 mM, preferably in
the range of from 0.1-500 mM.
Halide Sources
According to the invention the halide source needed
for the reaction with the haloperoxidase may be achieved in
many different ways, e.g., by adding a halide salt: It may be
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19
sodium chloride, potassium chloride, sodium bromide, potassium
bromide, sodium iodide, or potassium iodide.
The concentration of the halide source will typically
correspond to 0.01-1000 mM, preferably in the range of from
0.1-500 mM.
The composition
The composition comprising the haloperoxidase, the
hydrogen peroxide source, and the the halide source may be
formulated as a solid or a liquid, in particular in the form of
a non-dusting granulate, or a stabilised liquid.
When formulated as a solid all components may be
mixed together, e.g., as a powder, a granulate or a gelled
product.
When other than dry form compositions are used and
even in that case, it is preferred to use a two part
formulation system having the hydrogen peroxide separate from
the other components.
The composition of the invention may further comprise
auxiliary agents such as wetting agents, thickening agents,
buffer, stabilisers, perfume, colourants, fillers and the
like.
Useful wetting agents are surfactants, i.e., non-ionic,
anionic, amphoteric or zwitterionic surfactants.
The composition of the invention may be a concentrated
product or a ready-to-use product.
Haloperoxidase activity
According to the present invention haloperoxidase
activity may be measured as described in WO 97/04102, p. 13 1.
7-19.
The present invention is further illustrated in the
following examples which are not in any way intended to limit
the scope of the invention as claimed.
ERA~LE 1
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Homology building of the Curvularia verruculosa haloperoxidase
3D-structure
Using sequence homology of Curvularia ineaqualis (CI) to
5 other sequences, e.g., Curvularia verruculosa, CI-like 3 D
structures can be found.
In comparison with the Curvularia ineaqualis, used for
elucidating the structure, Curvularia verruculosa differs in a
number of residues. The model may be built using the INSIGHT
10 and HOMOLOGY programs from Molecular Simulations Inc. The
program substitutes the amino acids in the Curvularia
ineaqualis with amino acids from Curvularia verruculosa in the
homologous positions defined in the program as structurally
conserved regions (SCR). The residues in between are built
15 using the LOOP option with GENERATE. Using these steps a crude
model may be obtained which gives information of spatial
interactions.
The structure can be refined using the method described in
the HOMOLOGY package.
EXAMPLE 2
Construction of haloperoxidase variants
For the construction of variants of the Curvularia
verruculosa haloperoxidase enzyme the commercial kit,
Chameleon double-stranded, site-directed mutagenesis kit, can
be used according to the manufacturer's instructions.
The gene encoding the haloperoxidase enzyme in question
is located on the plasmid pElo29. In accordance with the
manufacturer's instructions, the ScaI site of the Ampicillin
gene of pElo29 is changed to a Mlul site by use of the primer
117996 (SEQ ID N0: 3). At the same time the desired mutation
is introduced into the haloperoxidase gene by the addition of
an appropriate primer comprising the desired mutation.
3S
Construction of the plasmid pElo29
The plasmid pElo29 was built from the following
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21
fragments:
a)The 4.1 kb fragment from the vector pCiP (described in
WO 93/34618) cut with BamHI and BglII.
b)The Curvularia verruculosa haloperoxidase gene
amplified by PCR with Pwo polymerase using the plasmid
pAJ014-1 (WO 97/04102) as template, and the primers
146063 and 146062 (SEQ ID N0: 4 and 5) introducing
BamHI and BgIII sites 5' and 3' respectively to the
haloperoxidase gene.
Following ligation of the fragments a and b, the whole
haloperoxidase gene were sequenced to ensure that no undesired
mutations had been introduced during the PCR amplification.
Site-directed mutagenesis
Site directed mutagenesis as described above was used to
construct plasmids harboring genes encoding variants of the
haloperoxidase enzyme. The following primers were used:
The primer 147293 (SEQ ID N0: 6) was used to introduce
D289L.
The primer 147295 (SEQ ID N0: 7) was used to introduce
D289E.
The primer 139078 (SEQ ID NO: 8) was used to introduce
R487E.
The primer 139085 (SEQ ID NO: 9) was used to introduce
V492S.
The mutations were in each case verified by sequencing
of the whole gene. The resulting plasmids were called pElo30,
pElo3l, pElo34 and pElo37 respectively.
Transformation of pElo30, pElo3l, pElo34 and pElo37 into JaL
228
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22
Aspergillus oryzae JaL 228 (WO 97/27221) is Aspergillus
oryzae IFO 4177 deleted in the alkaline protease and the
neutral metalloprotease I. This strain was transformed with
pElo30, pElo3l, pElo34 and pElo37 using the plasmid pToC90 (WO
91/17243) carrying the amdS gene as cotransformant. Selection
of transformants were performed using acetamide as described
in patent EP 0 531 372 B1. Transformants were spore reisolated
twice. Spores from second reisolation of each transformant
were tested for haloperoxidase production in small scale
fermentations (shake flasks and microtiterdishes).
Fermentation of Curvularia verruculosa haloperoxidase variants
in Aspergillus oryzae
Isolates from above were fermented in a tank medium
comprised of 12 g of sucrose, 20 g of 50~ yeast extract, 2 g
of MgS04*7H20, 2 g of KH2P04, 3 g of K2S04, 4 g of citric
acid, 1 ml of pluronic, 182 mg of V205, and 0.5 ml of trace
metals solutions per litre, and fed during the course of the
fermentation with a medium comprised of 250 g 80$ maltose
solution, 20 g of 50~ yeast extract, 5 g of citric acid, 5 ml
of pluronic, 182 mg of V205, and 0.5 ml of trace metals
solutions per litre. The fermentation broth were harvested
after 5 days of fermentation at 34oC, pH above 6Ø
~ Trace metals solution: 14.3 g of ZnS04*7H20, 2.5 g of
CuS04*5H20, 0.5 g of NiCl2*6H20, 13.8 g of FeS04*7H20, 8.5 g
of MnS04*H20, and 3.0 g of citric acid per litre.
Sequenc~ list
SEQ ID NO 3: Primer 117996; changed nucleotides are underlined
and leads to elimination of the Scal site, and introduction of
the Mlul site in pElo29:
5'-GA ATG ACT TGG TTG ACG CGT CAC CAG TCA C-3'
SEQ ID NO 4: Primer 146063; PCR primer for amplification of
the Curvularia verruculosa haloperoxidase gene. Underlined
nucleotides introduces the BamXI site:
5'-CGC GGA TCC TCT ATA TAC ACA ACT GG-3'
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23
SEQ ID NO 5: Primer 146062; PCR primer for amplification of
the Curvularia verruculosa haloperoxidase gene. Underlined
nucleotides introduces the BglII site:
5'-GAA GAT CTC GAG TTA ATT AAT CAC TGG-3'
l0 SEQ ID NO 6: Primer 147293; Underlined nucleotides introduces
the D289L mutation in the haloperoxidase enzyme in question:
5'-GG TCT GTA TTG GGC CTA CCT TGG GTC AAA CC-3'
20
SEQ ID NO 7: Primer 147295; Underlined nucleotides introduces
the D289E mutation in the haloperoxidase enzyme in question:
5'-GG TCT GTA TTG GGC CTA CGA GGG GTC AAA CC-3'
SEQ ID NO 8: Primer 139078; Underlined nucleotides introduces
the R487E mutation in the haloperoxidase enzyme in question:
5'-CG CCA TTT CTG AGA TCT TCC TGG GC-3'
SEQ ID NO 9: Primer 139085: Underlined nucleotides introduces
the V492S mutation in the haloperoxidase enzyme in question:
5'-GC ATC TTC CTC GGC AGC CAC TGG CGA TTC GAT GCC G-3'
EXA1~LE 3
pH-curves of various haloperoxidase variants
The haloperoxidase variants (derived from Curvularia
verruculosa) were made as described in Example 2.
Experimental:
Phenol red assay for pH-profile determination:
In 96 well microtiter plates:
100 ul 0.008$ phenol red in 60 mM Britton-Robinson buffer, pH
4-8.5. To these solutions were added 40 ul 0.5 M KBr and 50 ul
diluted enzyme solution containing 1 mM of ortho-vanadate. The
reaction was started by adding 10 ul 0.3~ hydrogen peroxide
and the kinetic was measured over 5 minutes at 595 nm.
Results:
Activity was taken relative to the highest value for each
enzyme:
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WO 99/47651 PCT/DK99/00133
24
ari- '~p'F~' per' per' pI~ p P P P P
ant 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
r v a u.ul u.14 n.a. u.ln u.34 1. . ~ v.~i v.io
wt
.U . U.
-
-
-
41 v ,Ty
U
~
It can be seen from the Table that the wild type has a pH
optimum at pH 7.0; the variant D289E and R487E and V492S have
a pH optimum at 6.0: and the variant D289L has a pH optimum at
pH 7.5.
EX»I~E 4
l0 pH-curve of purified haloperoxidase variant (V492S)
The haloperoxidase variant (V492S) described above and the
Curvularia verruculosa wild type were purified and tested with
Chicago Skye Blue:
Purification of haloperoxidases:
Fermentation broth containing haloperoxidase activity was
filtered GF/F (Whatmann) and 0.22 ~m (GS, Millipore) before
concentrating on the Filtron (cut off 10 kDa). The pH was
adjusted to pH 7.5 and the sample loaded onto a Q-Sepharose
column (Pharmacia) equilibrated in 50 mM Tris-HC1, pH 7.5. The
haloperoxidase was eluted in a linear gradient of 0-1 M NaCl in
50 mM Tris-HC1, pH 7.5. Haloperoxidase containing fractions
were concentrated on an Amicon cell (YM10 membrane) and loaded
onto MonoQ-column (Pharmicia) equilibrated in 50 mM Tris-HC1,
pH 8.5 and eluted in a linear gradient of 0-1 M NaCl in 50 mM
Tris-HC1. Haloperoxidase containing fractions were pooled and
further purified on a Superdex75 column 16/60 (Pharmacia)
equilibrated in 50 mM sodium acetate, 0.1 M NaCl, pH 5.5.
Experimental:
In 96 well microtiter plates:
100 P,1 60 mM Britton-Robinson pH 4-8 + 50 ~.1 enzyme solution +
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WO 99/47651 PCT/DK99/00133
25 ~,1 0.4 M NaCl + 25 ~1 Chicago Skye Blue diluted in water to
OD610 - 5. The reaction was started by adding 10 ~1 of 2 mM
H202. The activity was taken as the linear decrease in
adsorption at 595 nm.
5
Relative activities:
pH wt V492S
4 0 0.11
l0 4.5 0 0.62
5 0.25 0.93
5.5 1 1
6 0.89 0.70
6.5 0.41 0.34
15 7 0.11 0.08
7.5 0.02 0.02
8 0 0.01
Conclusion:
20 V492S clearly exhibits increased activity in the low pH range
compared to the wt enzyme.
30
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1
SEQUENCE LISTING
<110> NOVO NORDISK A/S
<120> HALOPEROXIDASES WITH ALTERED pH PROFILES
<130> 5516-WO
<140>
<141>
<160> 9
<170> PatentIn Ver. 2.0
<210> 1
<211> 609
<212> PRT
<213> Curvularia inaequalis
<400> 1
Met Gly Ser Val Thr Pro Ile Pro Leu Pro Lys Ile Asp Glu Pro Glu
1 5 10 15
Glu Tyr Asn Thr Asn Tyr Ile Leu Phe Trp Asn His Val Gly Leu Glu
20 25 30
Leu Asn Arg Val Thr His Thr Val Gly Gly Pro Leu Thr Gly Pro Pro
40 45
Leu Ser Ala Arg Ala Leu Gly Met Leu His Leu Ala Ile His Asp Ala
50 55 60
Tyr Phe Ser Ile Cys Pro Pro Thr Asp Phe Thr Thr Phe Leu Ser Pro
65 70 75 80
Asp Thr Glu Asn Ala Ala Tyr Arg Leu Pro Ser Pro Asn Gly Ala Asn
85 90 95
Asp Ala Arg Gln Ala Val Ala Gly Ala Ala Leu Lys Met Leu ser Ser
100 105 110
Leu Tyr Met Lys Pro Val Glu Gln Pro Asn Pro Asn Pro Gly Ala Asn
115 120 125
Ile Ser Asp Asn Ala Tyr Ala Gln Leu Gly Leu Val Leu Asp Arg Ser
130 135 140
Val Leu Glu Ala Pro Gly Gly Val Asp Arg Glu Ser Ala Ser Phe Met
145 150 155 160
Phe Gly Glu Asp Val Ala Asp Val Phe Phe Ala Leu Leu Asn Asp Pro
165 170 175
Arg Gly Ala Ser Gln Glu Gly Tyr His Pro Thr Pro Gly Arg Tyr Lys
180 185 190
Phe Asp Asp Glu Pro Thr His Pro Val Val Leu Ile Pro Val Asp Pro
195 200 205
Asn Asn Pro Asn Gly Pro Lys Met Pro Phe Arg Gln Tyr His Ala Pro
210 215 220
Phe Tyr Gly Lys Thr Thr Lys Arg Phe Ala Thr Gln Ser Glu His Phe
225 230 235 240
Leu Ala Asp Pro Pro Gly Leu Arg Ser Asn Ala Asp Glu Thr Ala Glu
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WO 99/47651 PCT/DK99/00133
2
245 250 255
Tyr Asp Asp Ala Val Arg Val Ala Ile Ala Met Gly Gly Ala Gln Ala
260 265 270
Leu Asn Ser Thr Lys Arg Ser Pro Trp Gln Thr Ala Gln Gly Leu Tyr
275 280 285
Trp Ala Tyr Asp Gly Ser Asn Leu Ile Gly Thr Pro Pro Arg Phe Tyr
290 295 300
Asn Gln Ile Val Arg Arg Ile Ala Val Thr Tyr Lys Lys Glu Glu Asp
305 310 315 320
Leu Ala Asn Ser Glu Val Asn Asn Ala Asp Phe Ala Arg Leu Phe Ala
325 330 335
Leu Val Asp Val Ala Cys Thr Asp Ala Gly Ile Phe Ser Trp Lys Glu
340 345 350
Lys Trp Glu Phe Glu Phe Trp Arg Pro Leu Ser Gly Val Arg Asp Asp
355 360 365
Gly Arg Pro Asp His Gly Asp Pro Phe Trp Leu Thr Leu Gly Ala Pro
370 375 380
Ala Thr Asn Thr Asn Asp Ile Pro Phe Lys Pro Pro Phe Pro Ala Tyr
385 390 395 400
Pro Ser Gly His Ala Thr Phe Gly Gly Ala Val Phe Gln Met Val Arg
405 410 415
Arg Tyr Tyr Asn Gly Arg Val Gly Thr Trp Lya Asp Asp Glu Pro Asp
420 425 430
Asn Ile Ala Ile Asp Met Met Ile Ser Glu Glu Leu Asn Gly Val Asn
435 440 445
Arg Asp Leu Arg Gln Pro Tyr Asp Pro Thr Ala Pro Ile Glu Asp Gln
450 455 460
Pro Gly Ile Val Arg Thr Arg Ile Val Arg His Phe Asp Ser Ala Trp
465 470 475 480
Glu Leu Met Phe Glu Asn Ala Ile Ser Arg Ile Phe Leu Gly Val His
485 490 495
Trp Arg Phe Asp Ala Ala Ala Ala Arg Asp IIe Leu Ile Pro Thr Thr
500 505 510
SQ
Thr Lys Asp Val Tyr Ala Val Asp Asn Asn Gly Ala Thr Val Phe Gln
515 520 525
Asn Val Glu Asp Ile Arg Tyr Thr Thr Arg Gly Thr Arg Glu Asp Pro
530 535 540
Glu Gly Leu Phe Pro Ile Gly Gly Val Pro Leu Gly Ile Glu Ile Ala
545 550 555 560
Asp Glu Ile Phe Asn Asn Gly Leu Lys Pro Thr Pro Pro Glu Ile Gln
565 570 575
Pro Met Pro Gln Glu Thr Pro Val Gln Lys Pro Val Gly Gln Gln Pro
580 585 590
Val Lys Gly Met Trp Glu Glu Glu Gln Ala Pro Val Val Lys Glu Ala
595 600 605
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WO 99/47651 PCT/DK99/00133
3
Pro
<210> 2
<211> 600
<212> PRT
<213> Curvularia sp.
<400> 2
Met Gly Ser Val Thr Pro Ile Pro Leu Pro Thr Ile Asp Glu Pro Glu
1 5 10 15
Glu Tyr Asn Asn Asn Tyr Ile Leu Phe Trp Asn Asn Val Gly Leu Glu
25 30
Leu Aan Arg Leu Thr His Thr Val Gly Gly Pro Leu Thr Gly Pro Pro
35 40 45
Leu Ser Ala Arg Ala Leu Gly Met Leu His Leu Ala Ile His Asp Ala
50 55 60
Tyr Phe Ser Ile Cys Pro Pro Thr Glu Phe Thr Thr Phe Leu Ser Pro
65 70 75 80
Asp Ala Glu Asn Pro Ala Tyr Arg Leu Pro Ser Pro Asn Gly Ala Asp
85 90 95
Asp Ala Arg Gln Ala Val Ala Gly Ala Ala Leu Lys Met Leu Ser Ser
100 105 110
Leu Tyr Met Lys Pro Ala Asp Pro Asn Thr Gly Thr Asn Ile Ser Asp
115 120 125
Asn Ala Tyr Ala Gln Leu Ala Leu Val Leu Glu Arg Ala Val Val Lys
130 135 140
Val Pro Gly Gly Val Asp Arg Glu Ser Val Ser Phe Met Phe Gly Glu
145 150 155 160
Ala Val Ala Asp Val Phe Phe Ala Leu Leu Asn Asp Pro Arg Gly Ala
165 170 175
Ser Gln Glu Gly Tyr Gln Pro Thr Pro Gly Arg Tyr Lys Phe Asp Asp
180 185 190
Glu Pro Thr His Pro Val Val Leu Val Pro Val Asp Pro Asn Asn Pro
195 200 205
Asn Gly Pro Lys Met Pro Phe Arg Gln Tyr His Ala Pro Phe Tyr Gly
210 215 220
Met Thr Thr Lys Arg Phe Ala Thr Gln Ser Glu His Ile Leu Ala Asp
225 230 235 240
Pro Pro Gly Leu Arg Ser Asn Ala Asp Glu Thr Ala Glu Tyr Asp Asp
245 250 255
Ser Ile Arg Val Ala Ile Ala Met Gly Gly Ala Gln Asp Leu Asn Ser
260 265 270
Thr Lys Arg Ser Pro Trp Gln Thr Ala Gln Gly Leu Tyr Trp AIa Tyr
275 280 285
Asp Gly Ser Asn Leu Val Gly Thr Pro Pro Arg Phe Tyr Asn Gln Ile
290 295 300
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WO 99/47651 PCT/DK99/00133
4
Val Arg Arg Ile Ala Val Thr Tyr Lys Lys Glu Asp Asp Leu Ala Asn
305 310 315 320
Ser Glu Val Asn Asn Ala Asp Phe Ala Arg Leu Phe Ala Leu Val Asn
325 330 335
Val Ala Cys Thr Asp Ala Gly Ile Phe Ser Trp Lys Glu Lys Trp Glu
340 345 350
Phe Glu Phe Trp Arg Pro Leu Ser Gly Val Arg Asp Asp Gly Arg Pro
355 360 365
Asp His Gly Asp Pro Phe Trp Leu Thr Leu Gly Ala Pro Ala Thr Asn
370 375 380
Thr Asn Asp Ile Pro Phe Lys Pro Pro Phe Pro Ala Tyr Pro Ser Gly
385 390 395 400
His Ala Thr Phe Gly Gly Ala Val Phe Gln Met Val Arg Arg Tyr Tyr
405 410 415
Asn Gly Arg Val Gly Thr Trp Lys Asp Asp Glu Pro Asp Asn Ile Ala
420 425 430
Ile Asp Met Met Ile Ser Glu Glu Leu Aan Gly Val Asn Arg Asp Leu
435 440 445
Arg Gln Pro Tyr Asp Pro Thr Ala Pro Ile Glu Asp Gln Pro Gly Ile
450 455 460
Val Arg Thr Arg Ile Val Arg His Phe Asp Ser Ala Trp Glu Met Met
465 470 475 480
Phe Glu Asn Ala Ile Ser Arg Ile Phe Leu Gly Val His Trp Arg Phe
485 490 495
Asp AIa Ala Ala Ala Arg Asp Ile Leu Ile Pro Thr Asn Thr Lys Asp
500 505 510
Val Tyr Ala Val Asp Ser Asn Gly Ala Thr Val Phe Gln Asn Val Glu
515 520 525
Asp Val Arg Tyr Ser Thr Lys Gly Thr Arg Glu Gly Arg Glu Gly Leu
530 535 540
Phe Pro Ile Gly Gly Val Pro Leu Gly Ile Glu Ile Ala Asp Glu Ile
545 550 555 560
Phe Asn Asn Gly Leu Arg Pro Thr Pro Pro Glu Leu Gln Pro Met Pro
565 570 575
Gln Asp Thr Pro Val Gln Lya Pro Val Gln GIy Met Trp Asp Glu Gln
580 585 590
Val Pro Leu Val Lys Glu Ala Pro
595 600
<2I0> 3
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
CA 02322781 2000-09-14
WO 99/47651 PC1'/DK99/00133
<400> 3
cgcggatcct ctatatacac aactgg 26
<210> 4
5 <211> 27
<212> DNA
<213> Artificial Sequence
<220>
10<223> Description of Artificial Primer
Sequence:
<400> 4
gaagatctcg agttaattaa tcactgg 27
15<210> 5
<211> 30
<212> DNA
<213> Artificial Sequence
20<220>
<223> Description of Artificial Primer
Sequence:
<400> 5
gaatgacttg gttgacgcgt caccagtcac 30
~
<210> 6
<211> 31
<2I2> DNA
<213> Artificial Sequence
30
<220>
<223> Description of Artificial Primer
Sequence:
<400> 6
35ggtctgtatt gggcctacct tgggtcaaac 31
c
<210> 7
<211> 31
<212> DNA
40<213> Artificial Sequence
<220>
<223> Description of Artificial Primer
Sequence:
45<400> 7
ggtctgtatt gggcctacga ggggtcaaac 31
c
<210> B
<211> 25
50< 212 > DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Primer
Sequence:
55
<400> 8
cgccatttct gagatcttcc tgggc 25
<210> 9
60<211> 36
<212> DNA
<213> Artificial Sequence
<220>
65<223> Description of Artificial Primer
Sequence:
<400> 9
CA 02322781 2000-09-14
WO 99/47651 PCT/DK99/00133
6
gcatcttcct cggcagccac tggcgattcg atgccg 36
