U.S. patent application number 09/832614 was filed with the patent office on 2002-05-16 for nucleic acids encoding polypeptides having haloperoxidase activity.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Danielsen, Steffen, Schneider, Palle.
Application Number | 20020058320 09/832614 |
Document ID | / |
Family ID | 27222377 |
Filed Date | 2002-05-16 |
United States Patent
Application |
20020058320 |
Kind Code |
A1 |
Danielsen, Steffen ; et
al. |
May 16, 2002 |
Nucleic acids encoding polypeptides having haloperoxidase
activity
Abstract
The present invention relates to isolated nucleic acid sequences
encoding polypeptides having haloperoxidase activity. The invention
also relates to nucleic acid constructs, vectors, and host cells
comprising the nucleic acid sequences.
Inventors: |
Danielsen, Steffen;
(Copenhagen, DK) ; Schneider, Palle; (Ballerup,
DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
C/O NOVO NORDISK OF NORTH AMERICA, INC.
405 LEXINGTON AVENUE, SUITE 6400
NEW YORK
NY
10174
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
27222377 |
Appl. No.: |
09/832614 |
Filed: |
April 11, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60202249 |
May 5, 2000 |
|
|
|
Current U.S.
Class: |
435/189 ;
435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12P 3/00 20130101; C12P
9/00 20130101; C11D 3/48 20130101; C11D 3/38654 20130101; C11D
3/38636 20130101; C12N 9/0065 20130101; C11D 3/3719 20130101 |
Class at
Publication: |
435/189 ;
435/325; 435/69.1; 435/320.1; 536/23.2 |
International
Class: |
C12N 009/02; C07H
021/04; C12P 021/02; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 14, 2000 |
DK |
PA 2000 00626 |
Claims
1. An isolated nucleic acid sequence comprising a nucleic acid
sequence which encodes a polypeptide having haloperoxidase
activity, wherein the polypeptide is selected from the group
consisting of: a) a polypeptide having an amino acid sequence which
has at least 80% homology with the amino acid sequence of SEQ ID
NO:2; b) a polypeptide which is encoded by a nucleic acid sequence
which hybridizes under medium stringency conditions with (i) the
nucleotide sequence of SEQ ID NO:1, (ii) a subsequence of (i) of at
least 100 nucleotides, or (iii) a complementary strand of (i) or
(ii); c) a variant of the polypeptide having an amino acid sequence
of SEQ ID NO:2 comprising a substitution, deletion, and/or
insertion of one or more amino acids; d) an allelic variant of (a)
or (b); e) a fragment of (a), (b), or (d) that has haloperoxidase
activity; and f) a polypeptide having more than 50% residual
activity after 15 minutes incubation at 70.degree. C and pH 7.
2. The nucleic acid sequence of claim 1, wherein the amino acid
sequence of the polypeptide has at least 95% homology with the
amino acid sequence of SEQ ID NO:2.
3. The nucleic acid sequence of any of claims 1-2, wherein the
polypeptide comprises the amino acid sequence of SEQ ID NO:2.
4. The nucleic acid sequence of any of claims 1-3, wherein the
polypeptide consists of the amino acid sequence of SEQ ID NO:2 or a
fragment thereof.
5. The nucleic acid sequence of claim 4, wherein the polypeptide
consists of the amino acid sequence of SEQ ID NO:2.
6. The nucleic acid sequence of claim 1, wherein the polypeptide is
a variant of the polypeptide having an amino acid sequence of SEQ
ID NO:2 comprising a substitution, deletion, and/or insertion of
one or more amino acids.
7. The nucleic acid sequence of claim 1, which is contained in the
plasmid contained in E. coli DH10B, deposited as DSM 13442.
8. An isolated nucleic acid sequence comprising a nucleic acid
sequence having at least one mutation in the polypeptide coding
sequence of SEQ ID NO:1, in which the mutant nucleic acid sequence
encodes a polypeptide consisting of the amino acid sequence of SEQ
ID NO:2.
10. A nucleic acid construct comprising the nucleic acid sequence
of any of claims 1-7 operably linked to one or more control
sequences that direct the production of the polypeptide in a
suitable expression host.
11. A recombinant expression vector comprising the nucleic acid
construct of claim 10.
12. A recombinant host cell comprising the nucleic acid construct
of claim 11.
13. A method for producing a mutant nucleic acid sequence,
comprising (a) introducing at least one mutation into the
polypeptide coding sequence of SEQ ID NO:1, wherein the mutant
nucleic acid sequence encodes a polypeptide consisting of the amino
acid sequence of SEQ ID NO:2; and (b) recovering the mutant nucleic
acid sequence.
14. A mutant nucleic acid sequence produced by the method of claim
13.
15. A method for producing a polypeptide, comprising (a)
cultivating a strain comprising the mutant nucleic acid sequence of
claim 14 encoding the polypeptide to produce a supernatant
comprising the polypeptide; and (b) recovering the polypeptide.
16. A method for producing the polypeptide of any of claims 1-7
comprising (a) cultivating a host cell comprising a nucleic acid
construct comprising a nucleic acid sequence encoding the
polypeptide under conditions suitable for production of the
polypeptide; and (b) recovering the polypeptide.
17. A method for producing a polypeptide comprising (a) cultivating
a host cell under conditions conducive for production of the
polypeptide, wherein the host cell comprises a mutant nucleic acid
sequence having at least one mutation in the polypeptide coding
sequence of SEQ ID NO:1, wherein the mutant nucleic acid sequence
encodes a polypeptide consisting of the amino acid sequence of SEQ
ID NO:2, and (b) recovering the polypeptide.
18. A method for shuffling of DNA comprising using the nucleotide
sequence of SEQ ID NO:1.
19. A polynucleotide encoding a polypeptide having haloperoxidase
activity obtainable by the method of claim 18.
20. A polypeptide having haloperoxidase activity encoded by the
polynucleotide of claim 19.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. 119 of
Danish application PA 2000 00626 filed Apr. 14, 2000, and U.S.
provisional application no. 60/202249, filed May 5, 2000 the
contents of which are fully incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to isolated nucleic acid
sequences encoding polypeptides having haloperoxidase activity. The
invention also relates to nucleic acid constructs, vectors, and
host cells comprising the nucleic acid sequences.
BACKGROUND
[0003] Haloperoxidases are widespread in nature being produced by
mammals, plants, algae, lichen, bacteria, and fungi.
Haloperoxidases are probably the enzymes responsible for the
formation of naturally occurring halogenated compounds. There are
three types of haloperoxidases, classified according to their
specificity for halide ions: Chloroperoxidases (E.C. 1.11.1.10)
which catalyze the chlorination, bromination and iodination of
compounds; bromoperoxidases which show specificity for bromide and
iodide ions; and iodoperoxidases (E.C. 1.11.1.8) which solely
catalyze the oxidation of iodide ions.
[0004] The first discovered haloperoxidases were determined to
contain heme as a prosthetic group or co-factor. However, more
recently, it has become apparent that there are numerous non-heme
haloperoxidases as well. Bacterial haloperoxidases have been found
with no prosthetic group. In addition, a number of other non-heme
haloperoxidases have been shown to possess a vanadium prosthetic
group. Haloperoxidases containing a vanadium prosthetic group are
known to include at least two types of fungal chloroperoxidases
from Curvularia inaequalis (van Schijndel et al., 1993, Biochimica
Biophysica Acta 1161:249-256; Simons et al., 1995, European Journal
of Biochemistry 229: 566-574; WO 95/27046) and Curvularia
verruculosa (WO 97/04102).
[0005] Haloperoxidases, like other oxidoreductases, are of current
interest because of their broad range of potential industrial
uses.
[0006] It is an object of the present invention to provide improved
polypeptides having haloperoxidase activity and nucleic acid
encoding the polypeptides.
SUMMARY OF THE INVENTION
[0007] The present invention relates to isolated nucleic acid
sequences encoding polypeptides having haloperoxidase activity
selected from the group consisting of:
[0008] (a) a polypeptide having an amino acid sequence which has at
least 80% homology with the amino acid sequence of SEQ ID NO:2;
[0009] (b) a polypeptide encoded by a nucleic acid sequence which
hybridizes under medium stringency conditions with (i) the
nucleotide sequence of SEQ ID NO:1, (ii) a subsequence of (i) of at
least 100 nucleotides, or (iii) a complementary strand of (i) or
(ii);
[0010] (c) a variant of the polypeptide having an amino acid
sequence of SEQ ID NO:2 comprising a substitution, deletion, and/or
insertion of one or more amino acids;
[0011] (d) an allelic variant of (a) or (b);
[0012] (e) a fragment of (a), (b), or (d) that has haloperoxidase
activity; and
[0013] (f) a polypeptide having more than 50% residual activity
after 15 minutes incubation at 70.degree. C. and pH 7.
[0014] The present invention also relates to nucleic acid
constructs, vectors, and host cells comprising the nucleic acid
sequences.
DETAILED DESCRIPTION OF THE INVENTION
[0015] Polypeptides Having Haloperoxidase Activity
[0016] The term "haloperoxidase activity" as defined herein
catalyzes the oxidation of a halide ion (X=Cl-, Br-, or I-) in the
presence of hydrogen peroxide (H.sub.2O.sub.2) to the corresponding
hypohalous acid (HOX):
H.sub.2O.sub.2+X-+H+->H.sub.2O+HOX
[0017] For purposes of the present invention, haloperoxidase
activity is determined according to the procedure described in
"Haloperoxidase assays" in the Examples.
[0018] In a first embodiment, the present invention relates to
isolated polypeptides having an amino acid sequence which has a
degree of homology to the amino acid sequence of SEQ ID NO:2 of at
least about 80%, preferably at least about 90%, more preferably at
least about 95%, and most preferably at least about 97%, which have
haloperoxidase activity (hereinafter "homologous polypeptides"). In
a preferred embodiment, the homologous polypeptides have an amino
acid sequence which differs by five amino acids, preferably by four
amino acids, more preferably by three amino acids, even more
preferably by two amino acids, and most preferably by one amino
acid from the amino acid sequence of SEQ ID NO:2. For purposes of
the present invention, the degree of homology between two amino
acid sequences is determined by using GAP version 8 from the GCG
package (Genetics Computer Group, 575 Science Drive, Madison, Wis.
53711, USA) with standard penalties for proteins: GAP weight 3.00,
length weight 0.100, Matrix described in Gribskov and Burgess,
Nucl. Acids Res. 14(16); 6745-6763 (1986).
[0019] Preferably, the polypeptides of the present invention
comprise the amino acid sequence of SEQ ID NO:2 or an allelic
variant thereof; or a fragment thereof that has haloperoxidase
activity. In a more preferred embodiment, the polypeptide of the
present invention comprises the amino acid sequence of SEQ ID NO:2.
In another preferred embodiment, the polypeptide of the present
invention consists of the amino acid sequence of SEQ ID NO:2 or an
allelic variant thereof; or a fragment thereof that has
haloperoxidase activity. In another preferred embodiment, the
polypeptide of the present invention consists of the amino acid
sequence of SEQ ID NO:2.
[0020] A fragment of SEQ ID NO:2 is a polypeptide having one or
more amino acids deleted from the amino and/or carboxyl terminus of
this amino acid sequence.
[0021] An allelic variant denotes any of two or more alternative
forms of a gene occupying the same chromosomal locus. Allelic
variation arises naturally through mutation, and may result in
polymorphism within populations. Gene mutations can be silent (no
change in the encoded polypeptide) or may encode polypeptides
having altered amino acid sequences. An allelic variant of a
polypeptide is a polypeptide encoded by an allelic variant of a
gene.
[0022] In a second embodiment, the present invention relates to
isolated polypeptides having haloperoxidase activity which are
encoded by nucleic acid sequences which hybridize under medium
stringency conditions, preferably medium-high stringency
conditions, more preferably high stringency conditions, and most
preferably very high stringency conditions with a nucleic acid
probe which hybridizes under the same conditions with (i) the
nucleotide sequence of SEQ ID NO:1, (ii) a subsequence of (i), or
(iii) a complementary strand of (i) or (ii) (J. Sambrook, E. F.
Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory
Manual, 2d edition, Cold Spring Harbor, New York). The subsequence
of SEQ ID NO:1 may be at least 100 nucleotides or preferably at
least 200 nucleotides. Moreover, the subsequence may encode a
polypeptide fragment, which has haloperoxidase activity. The
polypeptides may also be allelic variants or fragments of the
polypeptides that have haloperoxidase activity.
[0023] The nucleic acid sequence of SEQ ID NO:1 or a subsequence
thereof, as well as the amino acid sequence of SEQ ID NO:2 or a
fragment thereof, may be used to design a nucleic acid probe to
identify and clone DNA encoding polypeptides having haloperoxidase
activity from strains of different genera or species according to
methods well known in the art. In particular, such probes can be
used for hybridization with the genomic or cDNA of the genus or
species of interest, following standard Southern blotting
procedures, in order to identify and isolate the corresponding gene
therein. Such probes can be considerably shorter than the entire
sequence, but should be at least 15, preferably at least 25, and
more preferably at least 35 nucleotides in length. Longer probes
can also be used. Both DNA and RNA probes can be used. The probes
are typically labeled for detecting the corresponding gene (for
example, with .sup.32P, .sup.3H, .sup.35S, biotin, or avidin). Such
probes are encompassed by the present invention.
[0024] Thus, a genomic DNA or cDNA library prepared from such other
organisms may be screened for DNA, which hybridizes with the probes
described above and which encodes a polypeptide having
haloperoxidase activity. Genomic or other DNA from such other
organisms may be separated by agarose or polyacrylamide gel
electrophoresis, or other separation techniques. DNA from the
libraries or the separated DNA may be transferred to and
immobilized on nitrocellulose or other suitable carrier material.
In order to identify a clone or DNA which is homologous with SEQ ID
NO:1 or a subsequence thereof, the carrier material is used in a
Southern blot. For purposes of the present invention, hybridization
indicates that the nucleic acid sequence hybridizes to a labeled
nucleic acid probe corresponding to the nucleic acid sequence shown
in SEQ ID NO:1, its complementary strand, or a subsequence thereof,
under low to very high stringency conditions. Molecules to which
the nucleic acid probe hybridizes under these conditions are
detected using X-ray film.
[0025] In a preferred embodiment, the nucleic acid probe is a
nucleic acid sequence which encodes the polypeptide of SEQ ID NO:2,
or a subsequence thereof. In another preferred embodiment, the
nucleic acid probe is SEQ ID NO:1. In another preferred embodiment,
the nucleic acid probe is the nucleic acid sequence contained in
the pUC19 derived plasmid contained in Escherichia coli DH10B,
deposited as DSM 13442, wherein the nucleic acid sequence encodes a
polypeptide having haloperoxidase activity.
[0026] For long probes of at least 100 nucleotides in length, low
to very high stringency conditions are defined as prehybridization
and hybridization at 42.degree. C. in 5.times. SSPE, 0.3% SDS, 200
.mu.g/ml sheared and denatured salmon sperm DNA, and either 25%
formamide for low stringencies, 35% formamide for medium and
medium-high stringencies, or 50% formamide for high and very high
stringencies, following standard Southern blotting procedures.
[0027] For long probes of at least 100 nucleotides in length, the
carrier material is finally washed three times each for 15 minutes
using 2.times.SSC, 0.2% SDS preferably at least at 50.degree. C.
(low stringency), more preferably at least at 55.degree. C. (medium
stringency), more preferably at least at 60.degree. C. (medium-high
stringency), even more preferably at least at 65.degree. C. (high
stringency), and most preferably at least at 70.degree. C. (very
high stringency).
[0028] For short probes which are about 15 nucleotides to about 70
nucleotides in length, stringency conditions are defined as
prehybridization, hybridization, and washing post-hybridization at
about 5.degree. C. to about 10.degree. C. below the calculated
T.sub.m using the calculation according to Bolton and McCarthy
(1962, Proceedings of the National Academy of Sciences USA 48:1390)
in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40,
1.times. Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM
sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per
ml following standard Southern blofting procedures.
[0029] For short probes which are about 15 nucleotides to about 70
nucleotides in length, the carrier material is washed once in
6.times. SSC plus 0.1% SDS for 15 minutes and twice each for 15
minutes using 6.times. SSC at 5.degree. C. to 10.degree. C. below
the calculated T.sub.m.
[0030] In a third embodiment, the present invention relates to
variants of the polypeptide having an amino acid sequence of SEQ ID
NO:2 comprising a substitution, deletion, and/or insertion of one
or more amino acids.
[0031] The amino acid sequences of the variant polypeptides may
differ from the amino acid sequence of SEQ ID NO:2 by an insertion
or deletion of one or more amino acid residues and/or the
substitution of one or more amino acid residues by different amino
acid residues. Preferably, amino acid changes are of a minor
nature, that is conservative amino acid substitutions that do not
significantly affect the folding and/or activity of the protein;
small deletions, typically of one to about 30 amino acids; small
amino- or carboxyl-terminal extensions, such as an amino-terminal
methionine residue; a small linker peptide of up to about 20-25
residues; or a small extension that facilitates purification by
changing net charge or another function, such as a poly-histidine
tract, an antigenic epitope or a binding domain.
[0032] Examples of conservative substitutions are within the group
of basic amino acids (arginine, lysine and histidine), acidic amino
acids (glutamic acid and aspartic acid), polar amino acids
(glutamine and asparagine), hydrophobic amino acids (leucine,
isoleucine and valine), aromatic amino acids (phenylalanine,
tryptophan and tyrosine), and small amino acids (glycine, alanine,
serine, threonine and methionine). Amino acid substitutions, which
do not generally alter the specific activity are known in the art
and are described, for example, by H. Neurath and R. L. Hill, 1979,
In, The Proteins, Academic Press, New York. The most commonly
occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,
Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro,
Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as
these in reverse.
[0033] In a fourth embodiment, the present invention relates to
isolated polypeptides having a residual activity of at least 50%
residual activity, preferably at least 60% residual activity after
15 minutes incubation at 70.degree. C. and pH 7. In a preferred
embodiment, the polypeptides of the invention retain at least 50%
residual activity, preferably at least 80% residual activity after
15 minutes incubation at 60.degree. C. and pH 7.
[0034] In a preferred embodiment, the polypeptides of the invention
contain a vanadium prosthetic group, and accordingly they are
vanadium haloperoxidases. In another preferred embodiment, the
polypeptides of the invention are chloroperoxidases.
[0035] In a fifth embodiment, the present invention relates to
isolated polypeptides having immunochemical identity or partial
immunochemical identity to the polypeptide having the amino acid
sequence of SEQ ID NO:2. The immunochemical properties are
determined by immunological cross-reaction identity tests by the
well-known Ouchterlony double immunodiffusion procedure.
Specifically, an antiserum containing polyclonal antibodies which
are immunoreactive or bind to epitopes of the polypeptide having
the amino acid sequence of SEQ ID NO:2 are prepared by immunizing
rabbits (or other rodents) according to the procedure described by
Harboe and Ingild, In N. H. Axelsen, J. Krll, and B. Weeks,
editors, A Manual of Quantitative Immunoelectrophoresis, Blackwell
Scientific Publications, 1973, Chapter 23, or Johnstone and Thorpe,
Immunochemistry in Practice, Blackwell Scientific Publications,
1982 (more specifically pages 27-31). A polypeptide having
immunochemical identity is a polypeptide, which reacts with the
antiserum in an identical fashion such as total fusion of
precipitates, identical precipitate morphology, and/or identical
electrophoretic mobility using a specific immunochemical technique.
A further explanation of immunochemical identity is described by
Axelsen, Bock, and Krll, In N. H. Axelsen, J. Krll, and B. Weeks,
editors, A Manual of Quantitative Immunoelectrophoresis, Blackwell
Scientific Publications, 1973, Chapter 10. A polypeptide having
partial immunochemical identity is a polypeptide, which reacts with
the antiserum in a partially identical fashion such as partial
fusion of precipitates, partially identical precipitate morphology,
and/or partially identical electrophoretic mobility using a
specific immunochemical technique. A further explanation of partial
immunochemical identity is described by Bock and Axelsen, In N. H.
Axelsen, J. Krll, and B. Weeks, editors, A Manual of Quantitative
Immunoelectrophoresis, Blackwell Scientific Publications, 1973,
Chapter 11.
[0036] The antibody may also be a monoclonal antibody. Monoclonal
antibodies may be prepared and used, e.g., according to the methods
of E. Harlow and D. Lane, editors, 1988, Antibodies, A Laboratory
Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
[0037] The polypeptides of the present invention have at least 20%,
preferably at least 40%, more preferably at least 60%, even more
preferably at least 80%, even more preferably at least 90%, and
most preferably at least 100% of the haloperoxidase activity of the
polypeptide of SEQ ID NO:2.
[0038] A polypeptide of the present invention may be obtained from
microorganisms of any genus. For purposes of the present invention,
the term "obtained from" as used herein in connection with a given
source shall mean that the polypeptide encoded by the nucleic acid
sequence is produced by the source or by a cell in which the
nucleic acid sequence from the source has been inserted. In a
preferred embodiment, the polypeptide is secreted
extracellularly.
[0039] A polypeptide of the present invention may be a bacterial
polypeptide. For example, the polypeptide may be a gram positive
bacterial polypeptide such as a Bacillus polypeptide, e.g., a
Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,
Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus
lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus
stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis
polypeptide; or a Streptomyces polypeptide, e.g., a Streptomyces
lividans or Streptomyces murinus polypeptide; or a gram negative
bacterial polypeptide, e.g., an E. coli or a Pseudomonas sp.
polypeptide.
[0040] A polypeptide of the present invention may be a fungal
polypeptide, and more preferably a yeast polypeptide such as a
Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces,
or Yarrowia polypeptide; or more preferably a filamentous fungal
polypeptide such as an Acremonium, Aspergillus, Aureobasidium,
Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor,
Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,
Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus,
Thielavia, Tolypocladium, or Trichoderma polypeptide.
[0041] In a preferred embodiment, the polypeptide is a
Saccharomyces carlsbergensis, Saccharomyces cerevisiae,
Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces
kluyveri, Saccharomyces norbensis or Saccharomyces oviformis
polypeptide.
[0042] In another preferred embodiment, the polypeptide is an
Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,
Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger,
Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis,
Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,
Fusarium graminum, Fusarium heterosporum, Fusarium negundi,
Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides,
Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides,
Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor
miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium
purpurogenum, Trichoderma harzianum, Trichoderma koningii,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma
viride polypeptide.
[0043] In a preferred embodiment, the polypeptide is a
Geniculosporium sp. polypeptide, more preferably a Geniculosporium
sp. haloperoxidase, and most preferably a Geniculosporium sp.
haloperoxidase encoded by the nucleic acid sequence contained in
the plasmid contained in E. coli DH10B, deposited as DSM 13442,
e.g., the polypeptide with the amino acid sequence of SEQ ID
NO:2.
[0044] It will be understood that for the aforementioned species,
the invention encompasses both the perfect and imperfect states,
and other taxonomic equivalents, e.g., anamorphs, regardless of the
species name by which they are known. Those skilled in the art will
readily recognize the identity of appropriate equivalents.
[0045] Strains of these species are readily accessible to the
public in a number of culture collections, such as the American
Type Culture Collection (ATCC), Deutsche Sammlung von
Mikroorganismen und Zelikulturen GmbH (DSMZ), Centraalbureau Voor
Schimmelcultures (CBS), and Agricultural Research Service Patent
Culture Collection, Northern Regional Research Center (NRRL).
[0046] Furthermore, such polypeptides may be identified and
obtained from other sources including microorganisms isolated from
nature (e.g., soil, composts, water, etc.) using the
above-mentioned probes. Techniques for isolating microorganisms
from natural habitats are well known in the art. The nucleic acid
sequence may then be derived by similarly screening a genomic or
cDNA library of another microorganism. Once a nucleic acid sequence
encoding a polypeptide has been detected with the probe(s), the
sequence may be isolated or cloned by utilizing techniques which
are known to those of ordinary skill in the art (see, e.g.,
Sambrook et aL, 1989, supra).
[0047] As defined herein, an "isolated" polypeptide is a
polypeptide which is essentially free of other non-haloperoxidase
polypeptides, e.g., at least about 20% pure, preferably at least
about 40% pure, more preferably about 60% pure, even more
preferably about 80% pure, most preferably about 90% pure, and even
most preferably about 95% pure, as determined by SDS-PAGE.
[0048] Polypeptides encoded by nucleic acid sequences of the
present invention also include fused polypeptides or cleavable
fusion polypeptides in which another polypeptide is fused at the
N-terminus or the C-terminus of the polypeptide or fragment
thereof. A fused polypeptide is produced by fusing a nucleic acid
sequence (or a portion thereof) encoding another polypeptide to a
nucleic acid sequence (or a portion thereof) of the present
invention. Techniques for producing fusion polypeptides are known
in the art, and include ligating the coding sequences encoding the
polypeptides so that they are in frame and that expression of the
fused polypeptide is under control of the same promoter(s) and
terminator.
[0049] Nucleic Acid Sequences
[0050] The present invention also relates to isolated nucleic acid
sequences, which encode a polypeptide of the present invention. In
a preferred embodiment, the nucleic acid sequence is set forth in
SEQ ID NO:1. In another more preferred embodiment, the nucleic acid
sequence is the sequence contained in the pUC19 derived plasmid
contained in Escherichia coli DH10B, deposited as DSM 13442. The
present invention also encompasses nucleic acid sequences which
encode a polypeptide having the amino acid sequence of SEQ ID NO:2,
which differ from SEQ ID NO:1 by virtue of the degeneracy of the
genetic code. The present invention also relates to subsequences of
SEQ ID NO:1 which encode fragments of SEQ ID NO:2 that have
haloperoxidase activity.
[0051] A subsequence of SEQ ID NO:1 is a nucleic acid sequence
encompassed by SEQ ID NO:1 except that one or more nucleotides from
the 5' and/or 3' end have been deleted.
[0052] The present invention also relates to mutant nucleic acid
sequences comprising at least one mutation in the polypeptide
coding sequence of SEQ ID NO:1, in which the mutant nucleic acid
sequence encodes a polypeptide which consists of the amino acid
sequence of SEQ ID NO:2.
[0053] The techniques used to isolate or clone a nucleic acid
sequence encoding a polypeptide are known in the art and include
isolation from genomic DNA, preparation from cDNA, or a combination
thereof. The cloning of the nucleic acid sequences of the present
invention from such genomic DNA can be effected, e.g., by using the
well known polymerase chain reaction (PCR) or antibody screening of
expression libraries to detect cloned DNA fragments with shared
structural features. See, e.g., Innis et al., 1990, PCR: A Guide to
Methods and Application, Academic Press, New York. Other nucleic
acid amplification procedures such as ligase chain reaction (LCR),
ligated activated transcription (LAT) and nucleic acid
sequence-based amplification (NASBA) may be used. The nucleic acid
sequence may be cloned from a strain of Geniculosporium, or another
or related organism and thus, for example, may be an allelic or
species variant of the polypeptide encoding region of the nucleic
acid sequence.
[0054] The term "isolated nucleic acid sequence" as used herein
refers to a nucleic acid sequence which is essentially free of
other nucleic acid sequences, e.g., at least about 20% pure,
preferably at least about 40% pure, more preferably at least about
60% pure, even more preferably at least about 80% pure, and most
preferably at least about 90% pure as determined by agarose
electrophoresis. For example, an isolated nucleic acid sequence can
be obtained by standard cloning procedures used in genetic
engineering to relocate the nucleic acid sequence from its natural
location to a different site where it will be reproduced. The
cloning procedures may involve excision and isolation of a desired
nucleic acid fragment comprising the nucleic acid sequence encoding
the polypeptide, insertion of the fragment into a vector molecule,
and incorporation of the recombinant vector into a host cell where
multiple copies or clones of the nucleic acid sequence will be
replicated. The nucleic acid sequence may be of genomic, cDNA, RNA,
semisynthetic, synthetic origin, or any combinations thereof.
[0055] The present invention also relates to nucleic acid sequences
which have a degree of homology to the polypeptide coding sequence
of SEQ ID NO:1 of at least about 80%, preferably about 90%, more
preferably about 95%, and most preferably about 97% homology, which
encode an active polypeptide. For purposes of the present
invention, the degree of homology between two nucleic acid
sequences is determined by using GAP version 8 from the GCG package
with standard penalties for DNA: GAP weight 5.00, length weight
0.300, Matrix described in Gribskov and Burgess, Nucl. Acids Res.
14(16); 6745-6763 (1986).
[0056] Modification of a nucleic acid sequence encoding a
polypeptide of the present invention may be necessary for the
synthesis of polypeptides substantially similar to the polypeptide.
The term "substantially similar" to the polypeptide refers to
non-naturally occurring forms of the polypeptide. These
polypeptides may differ in some engineered way from the polypeptide
isolated from its native source, e.g., variants that differ in
specific activity, thermostability, pH optimum, or the like. The
variant sequence may be constructed on the basis of the nucleic
acid sequence presented as the polypeptide encoding part of SEQ ID
NO:1, e.g., a subsequence thereof, and/or by introduction of
nucleotide substitutions which do not give rise to another amino
acid sequence of the polypeptide encoded by the nucleic acid
sequence, but which correspond to the codon usage of the host
organism intended for production of the enzyme, or by introduction
of nucleotide substitutions which may give rise to a different
amino acid sequence. For a general description of nucleotide
substitution, see, e.g., Ford et al., 1991, Protein Expression and
Purification 2: 95-107.
[0057] It will be apparent to those skilled in the art that such
substitutions can be made outside the regions critical to the
function of the molecule and still result in an active polypeptide.
Amino acid residues essential to the activity of the polypeptide
encoded by the isolated nucleic acid sequence of the invention, and
therefore preferably not subject to substitution, may be identified
according to procedures known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham
and Wells, 1989, Science 244: 1081-1085). In the lafter technique,
mutations are introduced at every positively charged residue in the
molecule, and the resultant mutant molecules are tested for
haloperoxidase activity to identify amino acid residues that are
critical to the activity of the molecule. Sites of substrate-enzyme
interaction can also be determined by analysis of the
three-dimensional structure as determined by such techniques as
nuclear magnetic resonance analysis, crystallography or
photoaffinity labelling (see, e.g., de Vos et al., 1992, Science
255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224:
899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).
[0058] The present invention also relates to isolated nucleic acid
sequences encoding a polypeptide of the present invention, which
hybridize under low stringency conditions, preferably medium
stringency conditions, more preferably medium-high stringency
conditions, even more preferably high stringency conditions, and
most preferably very high stringency conditions with a nucleic acid
probe which hybridizes under the same conditions with the nucleic
acid sequence of SEQ ID NO:1 or its complementary strand; or
allelic variants and subsequences thereof (Sambrook et al., 1989,
supra), as defined herein.
[0059] The present invention also relates to isolated nucleic acid
sequences produced by (a) hybridizing a DNA under low, medium,
medium-high, high, or very high stringency conditions with (i) the
nucleotide sequence of SEQ ID NO:1, (ii) a subsequence of (i), or
(iii) a complementary strand of (i), (ii) or (iii); and (b)
isolating the nucleic acid sequence. The subsequence is preferably
a sequence of at least 100 nucleotides such as a sequence, which
encodes a polypeptide fragment which has haloperoxidase
activity.
[0060] Methods for Producing Mutant Nucleic Acid Sequences
[0061] The present invention further relates to methods for
producing a mutant nucleic acid sequence, comprising introducing at
least one mutation into the polypeptide coding sequence of SEQ ID
NO:1 or a subsequence thereof, wherein the mutant nucleic acid
sequence encodes a polypeptide which consists of the amino acid
sequence of SEQ ID NO:2 or a fragment thereof which has
haloperoxidase activity.
[0062] The introduction of a mutation into the nucleic acid
sequence to exchange one nucleotide for another nucleotide may be
accomplished by site-directed mutagenesis using any of the methods
known in the art. Particularly useful is the procedure, which
utilizes a supercoiled, double stranded DNA vector with an insert
of interest and two synthetic primers containing the desired
mutation. The oligonucleotide primers, each complementary to
opposite strands of the vector, extend during temperature cycling
by means of Pfu DNA polymerase. On incorporation of the primers, a
mutated plasmid containing staggered nicks is generated. Following
temperature cycling, the product is treated with Dpnl which is
specific for methylated and hemimethylated DNA to digest the
parental DNA template and to select for mutation-containing
synthesized DNA. Other procedures known in the art may also be
used.
[0063] Nucleic Acid Constructs
[0064] The present invention also relates to nucleic acid
constructs comprising a nucleic acid sequence of the present
invention operably linked to one or more control sequences, which
direct the expression of the coding sequence in a suitable host
cell under conditions compatible with the control sequences.
Expression will be understood to include any step involved in the
production of the polypeptide including, but not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, and secretion.
[0065] "Nucleic acid construct" is defined herein as a nucleic acid
molecule, either single- or double-stranded, which is isolated from
a naturally occurring gene or which has been modified to contain
segments of nucleic acid combined and juxtaposed in a manner that
would not otherwise exist in nature. The term nucleic acid
construct is synonymous with the term expression cassette when the
nucleic acid construct contains all the control sequences required
for expression of a coding sequence of the present invention. The
term "coding sequence" is defined herein as a nucleic acid
sequence, which directly specifies the amino acid sequence of its
protein product. The boundaries of a genomic coding sequence are
generally determined by a ribosome binding site (prokaryotes) or by
the ATG start codon (eukaryotes) located just upstream of the open
reading frame at the 5' end of the mRNA and a transcription
terminator sequence located just downstream of the open reading
frame at the 3' end of the mRNA. A coding sequence can include, but
is not limited to, DNA, cDNA, and recombinant nucleic acid
sequences.
[0066] An isolated nucleic acid sequence encoding a polypeptide of
the present invention may be manipulated in a variety of ways to
provide for expression of the polypeptide. Manipulation of the
nucleic acid sequence prior to its insertion into a vector may be
desirable or necessary depending on the expression vector. The
techniques for modifying nucleic acid sequences utilizing
recombinant DNA methods are well known in the art.
[0067] The term "control sequences" is defined herein to include
all components, which are necessary or advantageous for the
expression of a polypeptide of the present invention. Each control
sequence may be native or foreign to the nucleic acid sequence
encoding the polypeptide. Such control sequences include, but are
not limited to, a leader, polyadenylation sequence, propeptide
sequence, promoter, signal peptide sequence, and transcription
terminator. At a minimum, the control sequences include a promoter,
and transcriptional and translational stop signals. The control
sequences may be provided with linkers for the purpose of
introducing specific restriction sites facilitating ligation of the
control sequences with the coding region of the nucleic acid
sequence encoding a polypeptide. The term "operably linked" is
defined herein as a configuration in which a control sequence is
appropriately placed at a position relative to the coding sequence
of the DNA sequence such that the control sequence directs the
expression of a polypeptide.
[0068] The control sequence may be an appropriate promoter
sequence, a nucleic acid sequence that is recognized by a host cell
for expression of the nucleic acid sequence. The promoter sequence
contains transcriptional control sequences, which mediate the
expression of the polypeptide. The promoter may be any nucleic acid
sequence which shows transcriptional activity in the host cell of
choice including mutant, truncated, and hybrid promoters, and may
be obtained from genes encoding extracellular or intracellular
polypeptides either homologous or heterologous to the host
cell.
[0069] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention, especially in a bacterial host cell, are the promoters
obtained from the E. coli fac operon, Streptomyces coelicolor
agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),
Bacillus licheniformis alpha-amylase gene (amyL), Bacillus
stearothermophilus maltogenic amylase gene (amyM), Bacillus
amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis
penicillinase gene (penP), Bacillus subtilis xyIA and xyIB genes,
and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,
Proceedings of the National Academy of Sciences USA 75: 3727-3731),
as well as the tac promoter (DeBoer et al., 1983, Proceedings of
the National Academy of Sciences USA 80: 21-25). Further promoters
are described in "Useful proteins from recombinant bacteria" in
Scientific American, 1980, 242: 74-94; and in Sambrook et al.,
1989, supra.
[0070] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention in a filamentous fungal host cell are promoters obtained
from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor
miehei aspartic proteinase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,
Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans
acetamidase, and Fusarium oxysporum trypsin-like protease (WO
96/00787), as well as the NA2-tpi promoter (a hybrid of the
promoters from the genes for Aspergillus niger neutral
alpha-amylase and Aspergillus oryzae triose phosphate isomerase),
and mutant, truncated, and hybrid promoters thereof.
[0071] In a yeast host, useful promoters are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP),
and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other
useful promoters for yeast host cells are described by Romanos et
al., 1992, Yeast 8: 423-488.
[0072] The control sequence may also be a suitable transcription
terminator sequence, a sequence recognized by a host cell to
terminate transcription. The terminator sequence is operably linked
to the 3' terminus of the nucleic acid sequence encoding the
polypeptide. Any terminator which is functional in the host cell of
choice may be used in the present invention.
[0073] Preferred terminators for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase,
Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate
synthase, Aspergillus niger alpha-glucosidase, and Fusarium
oxysporum trypsin-like protease.
[0074] Preferred terminators for yeast host cells are obtained from
the genes for Saccharomyces cerevisiae enolase, Saccharomyces
cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae
glyceraldehyde-3-phosph- ate dehydrogenase. Other useful
terminators for yeast host cells are described by Romanos et al.,
1992, supra.
[0075] The control sequence may also be a suitable leader sequence,
a nontranslated region of an mRNA which is important for
translation by the host cell. The leader sequence is operably
linked to the 5' terminus of the nucleic acid sequence encoding the
polypeptide. Any leader sequence that is functional in the host
cell of choice may be used in the present invention.
[0076] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase.
[0077] Suitable leaders for yeast host cells are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae
alpha-factor, and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH2/GAP).
[0078] The control sequence may also be a polyadenylation sequence,
a sequence operably linked to the 3' terminus of the nucleic acid
sequence and which, when transcribed, is recognized by the host
cell as a signal to add polyadenosine residues to transcribed mRNA.
Any polyadenylation sequence which is functional in the host cell
of choice may be used in the present invention.
[0079] Preferred polyadenylation sequences for filamentous fungal
host cells are obtained from the genes for Aspergillus oryzae TAKA
amylase, Aspergillus niger glucoamylase, Aspergillus nidulans
anthranilate synthase, Fusarium oxysporum trypsin-like protease,
and Aspergillus niger alpha-glucosidase.
[0080] Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Molecular Cellular Biology 15:
5983-5990.
[0081] The control sequence may also be a signal peptide coding
region that codes for an amino acid sequence linked to the amino
terminus of a polypeptide and directs the encoded polypeptide into
the cell's secretory pathway. The 5' end of the coding sequence of
the nucleic acid sequence may inherently contain a signal peptide
coding region naturally linked in translation reading frame with
the segment of the coding region which encodes the secreted
polypeptide. Alternatively, the 5' end of the coding sequence may
contain a signal peptide coding region which is foreign to the
coding sequence. The foreign signal peptide coding region may be
required where the coding sequence does not naturally contain a
signal peptide coding region. Alternatively, the foreign signal
peptide coding region may simply replace the natural signal peptide
coding region in order to enhance secretion of the polypeptide.
However, any signal peptide coding region which directs the
expressed polypeptide into the secretory pathway of a host cell of
choice may be used in the present invention.
[0082] Effective signal peptide coding regions for bacterial host
cells are the signal peptide coding regions obtained from the genes
for Bacillus NCIB 11837 maltogenic amylase, Bacillus
stearothermophilus alpha-amylase, Bacillus licheniformis
subtilisin, Bacillus licheniformis beta-lactamase, Bacillus
stearothermophilus neutral proteases (nprT, nprS, nprM), and
Bacillus subtilis prsA. Further signal peptides are described by
Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
[0083] Effective signal peptide coding regions for filamentous
fungal host cells are the signal peptide coding regions obtained
from the genes for Aspergillus oryzae TAKA amylase, Aspergillus
niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor
miehei aspartic proteinase, Humicola insolens cellulase, and
Humicola lanuginosa lipase.
[0084] Useful signal peptides for yeast host cells are obtained
from the genes for Saccharomyces cerevisiae alpha-factor and
Saccharomyces cerevisiae invertase. Other useful signal peptide
coding regions are described by Romanos et al., 1992, supra.
[0085] The control sequence may also be a propeptide coding region
that codes for an amino acid sequence positioned at the amino
terminus of a polypeptide. The resultant polypeptide is known as a
proenzyme or propolypeptide (or a zymogen in some cases). A
propolypeptide is generally inactive and can be converted to a
mature active polypeptide by catalytic or autocatalytic cleavage of
the propeptide from the propolypeptide. The propeptide coding
region may be obtained from the genes for Bacillus subtilis
alkaline protease (aprE), Bacillus subtilis neutral protease
(nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei
aspartic proteinase, and Myceliophthora thermophila laccase (WO
95/33836).
[0086] Where both signal peptide and propeptide regions are present
at the amino terminus of a polypeptide, the propeptide region is
positioned next to the amino terminus of a polypeptide and the
signal peptide region is positioned next to the amino terminus of
the propeptide region.
[0087] It may also be desirable to add regulatory sequences, which
allow the regulation of the expression of the polypeptide relative
to the growth of the host cell. Examples of regulatory systems are
those which cause the expression of the gene to be turned on or off
in response to a chemical or physical stimulus, including the
presence of a regulatory compound. Regulatory systems in
prokaryotic systems include the lac, tac, and trp operator systems.
In yeast, the ADH2 system or GALL system may be used. In
filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus
niger glucoamylase promoter, and Aspergillus oryzae glucoamylase
promoter may be used as regulatory sequences. Other examples of
regulatory sequences are those which allow for gene amplification.
In eukaryotic systems, these include the dihydrofolate reductase
gene, which is amplified in the presence of methotrexate, and the
metallothionein genes, which are amplified with heavy metals. In
these cases, the nucleic acid sequence encoding the polypeptide
would be operably linked with the regulatory sequence.
[0088] Expression Vectors
[0089] The present invention also relates to recombinant expression
vectors comprising a nucleic acid sequence of the present
invention, a promoter, and transcriptional and translational stop
signals. The various nucleic acid and control sequences described
above may be joined together to produce a recombinant expression
vector which may include one or more convenient restriction sites
to allow for insertion or substitution of the nucleic acid sequence
encoding the polypeptide at such sites. Alternatively, the nucleic
acid sequence of the present invention may be expressed by
inserting the nucleic acid sequence or a nucleic acid construct
comprising the sequence into an appropriate vector for expression.
In creating the expression vector, the coding sequence is located
in the vector so that the coding sequence is operably linked with
the appropriate control sequences for expression.
[0090] The recombinant expression vector may be any vector (e.g., a
plasmid or virus), which can be conveniently subjected to
recombinant DNA procedures and can bring about the expression of
the nucleic acid sequence. The choice of the vector will typically
depend on the compatibility of the vector with the host cell into
which the vector is to be introduced. The vectors may be linear or
closed circular plasmids.
[0091] 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, an extrachromosomal element, a minichromosome, or
an artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
which, when introduced into the host cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. Furthermore, a single vector or plasmid or two
or more vectors or plasmids which together contain the total DNA to
be introduced into the genome of the host cell, or a transposon may
be used.
[0092] The vectors of the present invention preferably contain one
or more selectable markers, which permit easy selection of
transformed cells. A selectable marker is a gene the product of
which provides for biocide or viral resistance, resistance to heavy
metals, prototrophy to auxotrophs, and the like. Examples of
bacterial selectable markers are the dal genes from Bacillus
subtilis or Bacillus licheniformis, or markers, which confer
antibiotic resistance such as ampicillin, kanamycin,
chloramphenicol or tetracycline resistance. Suitable markers for
yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
Selectable markers for use in a filamentous fungal host cell
include, but are not limited to, amdS (acetamidase), argB
(ornithine carbamoyltransferase), bar (phosphinothricin
acetyltransferase), hph (hygromycin phosphotransferase), niaD
(nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase),
sC (sulfate adenyltransferase), and trpC (anthranilate synthase),
as well as equivalents thereof. Preferred for use in an Aspergillus
cell are the amdS and pyrG genes of Aspergillus nidulans or
Aspergillus oryzae and the bar gene of Streptomyces
hygroscopicus.
[0093] The vectors of the present invention preferably contain an
element(s) that permits integration of the vector into the host
cell's genome or autonomous replication of the vector in the cell
independent of the genome.
[0094] For integration into the host cell genome, the vector may
rely on the nucleic acid sequence encoding the polypeptide or any
other element of the vector for integration of the vector into the
genome by homologous or nonhomologous recombination. Alternatively,
the vector may contain additional nucleic acid sequences for
directing integration by homologous recombination into the genome
of the host cell. The additional nucleic acid sequences enable the
vector to be integrated into the host cell genome at a precise
location(s) in the chromosome(s). To increase the likelihood of
integration at a precise location, the integrational elements
should preferably contain a sufficient number of nucleic acids,
such as 100 to 10,000 base pairs, preferably 400 to 10,000 base
pairs, and most preferably 800 to 10,000 base pairs, which are
highly homologous with the corresponding target sequence to enhance
the probability of homologous recombination. The integrational
elements may be any sequence that is homologous with the target
sequence in the genome of the host cell. Furthermore, the
integrational elements may be non-encoding or encoding nucleic acid
sequences. On the other hand, the vector may be integrated into the
genome of the host cell by non-homologous recombination.
[0095] For autonomous replication, the vector may further comprise
an origin of replication enabling the vector to replicate
autonomously in the host cell in question. Examples of bacterial
origins of replication are the origins of replication of plasmids
pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E.
coli, and pUB110, pE194, pTA1060, and pAM.beta.1 permitting
replication in Bacillus. Examples of origins of replication for use
in a yeast host cell are the 2 micron origin of replication, ARS1,
ARS4, the combination of ARSI and CEN3, and the combination of ARS4
and CEN6. The origin of replication may be one having a mutation
which makes its functioning temperature-sensitive in the host cell
(see, e.g., Ehrlich, 1978, Proceedings of the National Academy of
Sciences USA 75: 1433).
[0096] More than one copy of a nucleic acid sequence of the present
invention may be inserted into the host cell to increase production
of the gene product. An increase in the copy number of the nucleic
acid sequence can be obtained by integrating at least one
additional copy of the sequence into the host cell genome or by
including an amplifiable selectable marker gene with the nucleic
acid sequence where cells containing amplified copies of the
selectable marker gene, and thereby additional copies of the
nucleic acid sequence, can be selected for by cultivating the cells
in the presence of the appropriate selectable agent.
[0097] The procedures used to ligate the elements described above
to construct the recombinant expression vectors of the present
invention are well known to one skilled in the art (see, e.g.,
Sambrook et al., 1989, supra).
[0098] Host Cells
[0099] The present invention also relates to recombinant host
cells, comprising a nucleic acid sequence of the invention, which
are advantageously used in the recombinant production of the
polypeptides. A vector comprising a nucleic acid sequence of the
present invention is introduced into a host cell so that the vector
is maintained as a chromosomal integrant or as a self-replicating
extra-chromosomal vector as described earlier. The term "host cell"
encompasses any progeny of a parent cell that is not identical to
the parent cell due to mutations that occur during replication. The
choice of a host cell will to a large extent depend upon the gene
encoding the polypeptide and its source.
[0100] The host cell may be a unicellular microorganism, e.g., a
prokaryote, or a non-unicellular microorganism, e.g., a
eukaryote.
[0101] Useful unicellular cells are bacterial cells such as gram
positive bacteria including, but not limited to, a Bacillus cell,
e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus
brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,
Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus
megaterium, Bacillus stearothermophilus, Bacillus subtilis, and
Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces
lividans and Streptomyces murinus, or gram negative bacteria such
as E. coli and Pseudomonas sp. In a preferred embodiment, the
bacterial host cell is a Bacillus lentus, Bacillus licheniformis,
Bacillus stearothermophilus, or Bacillus subtilis cell. In another
preferred embodiment, the Bacillus cell is an alkalophilic
Bacillus.
[0102] The introduction of a vector into a bacterial host cell may,
for instance, be effected by protoplast transformation (see, e.g.,
Chang and Cohen, 1979, Molecular General Genetics 168: 111-115),
using competent cells (see, e.g., Young and Spizizin, 1961, Journal
of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971,
Journal of Molecular Biology 56: 209-221), electroporation (see,
e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or
conjugation (see, e.g., Koehler and Thorne, 1987, Journal of
Bacteriology 169: 5771-5278).
[0103] The host cell may be a eukaryote, such as a mammalian,
insect, plant, or fungal cell.
[0104] In a preferred embodiment, the host cell is a fungal cell.
"Fungi" as used herein includes the phyla Ascomycota,
Basidiomycota, Chytridiomycota, and Zygomycota (as defined by
Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The
Fungi, 8th edition, 1995, CAB International, University Press,
Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et
al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et
aL, 1995, supra).
[0105] In a more preferred embodiment, the fungal host cell is a
yeast cell. "Yeast" as used herein includes ascosporogenous yeast
(Endomycetales), basidiosporogenous yeast, and yeast belonging to
the Fungi Imperfecti (Blastomycetes). Since the classification of
yeast may change in the future, for the purposes of this invention,
yeast shall be defined as described in Biology and Activities of
Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds,
Soc. App. Bacteriol. Symposium Series No. 9, 1980).
[0106] In an even more preferred embodiment, the yeast host cell is
a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,
Schizosaccharomyces, or Yarrowia cell.
[0107] In a most preferred embodiment, the yeast host cell is a
Saccharomyces carlsbergensis, Saccharomyces cerevisiae,
Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces
kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell.
In another most preferred embodiment, the yeast host cell is a
Kluyveromyces lactis cell. In another most preferred embodiment,
the yeast host cell is a Yarrowia lipolytica cell.
[0108] In another more preferred embodiment, the fungal host cell
is a filamentous fungal cell. "Filamentous fungi" include all
filamentous forms of the subdivision Eumycota and Oomycota (as
defined by Hawksworth et al., 1995, supra). The filamentous fungi
are generally characterized by a mycelial wall composed of chitin,
cellulose, glucan, chitosan, mannan, and other complex
polysaccharides. Vegetative growth is by hyphal elongation and
carbon catabolism is obligately aerobic. In contrast, vegetative
growth by yeasts such as Saccharomyces cerevisiae is by budding of
a unicellular thallus and carbon catabolism may be
fermentative.
[0109] In an even more preferred embodiment, the filamentous fungal
host cell is a cell of a species of, but not limited to,
Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora,
Neurospora, Penicillium, Thielavia, Tolypocladium, or
Trichoderma.
[0110] In a most preferred embodiment, the filamentous fungal host
cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus
japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus
oryzae cell. In another most preferred embodiment, the filamentous
fungal host cell is a Fusarium bactridioides, Fusarium cerealis,
Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,
Fusarium graminum, Fusarium heterosporum, Fusarium negundi,
Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides,
Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides,
or Fusarium venenatum cell. In an even most preferred embodiment,
the filamentous fungal parent cell is a Fusarium venenatum
(Nirenberg sp. nov.) cell. In another most preferred embodiment,
the filamentous fungal host cell is a Humicola insolens, Humicola
lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora
crassa, Penicillium purpurogenum, Thielavia terrestris, Trichoderma
harzianum, Trichoderma koningii, Trichoderma longibrachiatum,
Trichoderma reesei, or Trichoderma viride cell.
[0111] Fungal cells may be transformed by a process involving
protoplast formation, transformation of the protoplasts, and
regeneration of the cell wall in a manner known per se. Suitable
procedures for transformation of Aspergillus host cells are
described in EP 238 023 and Yelton et al., 1984, Proceedings of the
National Academy of Sciences USA 81: 1470-1474. Suitable methods
for transforming Fusarium species are described by Malardier et
al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be
transformed using the procedures described by Becker and Guarente,
In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast
Genetics and Molecular Biology, Methods in Enzymology, Volume 194,
pp 182-187, Academic Press, Inc., New York; Ito et al., 1983,
Journal of Bacteriology 153: 163; and Hinnen et al., 1978,
Proceedings of the National Academy of Sciences USA 75: 1920.
[0112] Methods of Production
[0113] The present invention also relates to methods for producing
a polypeptide of the present invention comprising (a) cultivating a
strain, which in its wild-type form is capable of producing the
polypeptide, to produce a supernatant comprising the polypeptide;
and (b) recovering the polypeptide. Preferably, the strain is of
the genus Geniculosporium.
[0114] The present invention also relates to methods for producing
a polypeptide of the present invention comprising (a) cultivating a
host cell under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
[0115] The present invention also relates to methods for producing
a polypeptide of the present invention comprising (a) cultivating a
host cell under conditions conducive for production of the
polypeptide, wherein the host cell comprises a mutant nucleic acid
sequence having at least one mutation in the polypeptide coding
region of SEQ ID NO:1, wherein the mutant nucleic acid sequence
encodes a polypeptide which consists of the amino acid sequence of
SEQ ID NO:2, and (b) recovering the polypeptide.
[0116] In the production methods of the present invention, the
cells are cultivated in a nutrient medium suitable for production
of the polypeptide using methods known in the art. For example, the
cell may be cultivated by shake flask cultivation, and small-scale
or large-scale fermentation (including continuous, batch,
fed-batch, or solid state fermentations) in laboratory or
industrial fermentors performed in a suitable medium and under
conditions allowing the polypeptide to be expressed and/or
isolated. The cultivation takes place in a suitable nutrient medium
comprising carbon and nitrogen sources and inorganic salts, using
procedures known in the art. Suitable media are available from
commercial suppliers or may be prepared according to published
compositions (e.g., in catalogues of the American Type Culture
Collection). If the polypeptide is secreted into the nutrient
medium, the polypeptide can be recovered directly from the medium.
If the polypeptide is not secreted, it can be recovered from cell
lysates.
[0117] The polypeptides may be detected using methods known in the
art that are specific for the polypeptides. These detection methods
may include use of specific antibodies, formation of an enzyme
product, or disappearance of an enzyme substrate. For example, an
enzyme assay may be used to determine the activity of the
polypeptide as described herein.
[0118] The resulting polypeptide may be recovered by methods known
in the art. For example, the polypeptide may be recovered from the
nutrient medium by conventional procedures including, but not
limited to, centrifugation, filtration, extraction, spray-drying,
evaporation, or precipitation.
[0119] The polypeptides of the present invention may be purified by
a variety of procedures known in the art including, but not limited
to, chromatography (e.g., ion exchange, affinity, hydrophobic,
chromatofocusing, and size exclusion), electrophoretic procedures
(e.g., preparative isoelectric focusing), differential solubility
(e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction
(see, e.g., Protein Purification, J.-C. Janson and Lars Ryden,
editors, VCH Publishers, New York, 1989).
[0120] Plants
[0121] The present invention also relates to a transgenic plant,
plant part, or plant cell, which has been transformed with a
nucleic acid sequence encoding a polypeptide having haloperoxidase
activity of the present invention so as to express and produce the
polypeptide in recoverable quantities. The polypeptide may be
recovered from the plant or plant part. Alternatively, the plant or
plant part containing the recombinant polypeptide may be used as
such for improving the quality of a food or feed, e.g., improving
nutritional value, palatability, and rheological properties, or to
destroy an antinutritive factor.
[0122] The transgenic plant can be dicotyledonous (a dicot) or
monocotyledonous (a monocot). Examples of monocot plants are
grasses, such as meadow grass (blue grass, Poa), forage grass such
as festuca, lolium, temperate grass, such as Agrostis, and cereals,
e.g., wheat, oats, rye, barley, rice, sorghum, and maize
(corn).
[0123] Examples of dicot plants are tobacco, legumes, such as
lupins, potato, sugar beet, pea, bean and soybean, and cruciferous
plants (family Brassicaceae), such as cauliflower, rape seed, and
the closely related model organism Arabidopsis thaliana.
[0124] Examples of plant parts are stem, callus, leaves, root,
fruits, seeds, and tubers. Also specific plant tissues, such as
chloroplast, apoplast, mitochondria, vacuole, peroxisomes, and
cytoplasm are considered to be a plant part. Furthermore, any plant
cell, whatever the tissue origin, is considered to be a plant
part.
[0125] Also included within the scope of the present invention are
the progeny of such plants, plant parts and plant cells.
[0126] The transgenic plant or plant cell expressing a polypeptide
of the present invention may be constructed in accordance with
methods known in the art. Briefly, the plant or plant cell is
constructed by incorporating one or more expression constructs
encoding a polypeptide of the present invention into the plant host
genome and propagating the resulting modified plant or plant cell
into a transgenic plant or plant cell.
[0127] Conveniently, the expression construct is a nucleic acid
construct, which comprises a nucleic acid sequence encoding a
polypeptide of the present invention operably linked with
appropriate regulatory sequences required for expression of the
nucleic acid sequence in the plant or plant part of choice.
Furthermore, the expression construct may comprise a selectable
marker useful for identifying host cells into which the expression
construct has been integrated and DNA sequences necessary for
introduction of the construct into the plant in question (the
latter depends on the DNA introduction method to be used).
[0128] The choice of regulatory sequences, such as promoter and
terminator sequences and optionally signal or transit sequences is
determined, for example, on the basis of when, where, and how the
polypeptide is desired to be expressed. For instance, the
expression of the gene encoding a polypeptide of the present
invention may be constitutive or inducible, or may be
developmental, stage or tissue specific, and the gene product may
be targeted to a specific tissue or plant part such as seeds or
leaves. Regulatory sequences are, for example, described by Tague
et al., 1988, Plant Physiology 86: 506.
[0129] For constitutive expression, the 35S-CaMV promoter may be
used (Franck et al., 1980, Cell 21: 285-294). Organ-specific
promoters may be, for example, a promoter from storage sink tissues
such as seeds, potato tubers, and fruits (Edwards & Coruzzi,
1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sink tissues
such as meristems (Ito et al., 1994, Plant Mol. Biol. 24: 863-878),
a seed specific promoter such as the glutelin, prolamin, globulin,
or albumin promoter from rice (Wu et aL., 1998, Plant and Cell
Physiology 39: 885-889), a Vicia faba promoter from the legumin B4
and the unknown seed protein gene from Vicia faba (Conrad et al.,
1998, Journal of Plant Physiology 152: 708-71 1), a promoter from a
seed oil body protein (Chen et al., 1998, Plant and Cell Physiology
39: 935-941), the storage protein napA promoter from Brassica
napus, or any other seed specific promoter known in the art, e.g.,
as described in WO 91/14772. Furthermore, the promoter may be a
leaf specific promoter such as the rbcs promoter from rice or
tomato (Kyozuka et al., 1993, Plant Physiology 102: 991-1000, the
chlorella virus adenine methyltransferase gene promoter (Mitra and
Higgins, 1994, Plant Molecular Biology 26: 85-93), or the aldP gene
promoter from rice (Kagaya et al., 1995, Molecular and General
Genetics 248: 668-674), or a wound inducible promoter such as the
potato pin2 promoter (Xu et al., 1993, Plant Molecular Biology 22:
573-588).
[0130] A promoter enhancer element may also be used to achieve
higher expression of the enzyme in the plant. For instance, the
promoter enhancer element may be an intron, which is placed between
the promoter and the nucleotide sequence encoding a polypeptide of
the present invention. For instance, Xu et al., 1993, supra
disclose the use of the first intron of the rice actin 1 gene to
enhance expression.
[0131] The selectable marker gene and any other parts of the
expression construct may be chosen from those available in the
art.
[0132] The nucleic acid construct is incorporated into the plant
genome according to conventional techniques known in the art,
including Agrobacterium-mediated transformation, virus-mediated
transformation, microinjection, particle bombardment, biolistic
transformation, and electroporation (Gasser et al., 1990, Science
244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al.,
1989, Nature 338: 274).
[0133] Presently, Agrobacterium tumefaciens-mediated gene transfer
is the method of choice for generating transgenic dicots (for a
review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology
19: 15-38). However it can also be used for transforming monocots,
although other transformation methods are generally preferred for
these plants. Presently, the method of choice for generating
transgenic monocots is particle bombardment (microscopic gold or
tungsten particles coated with the transforming DNA) of embryonic
calli or developing embryos (Christou, 1992, Plant Journal 2:
275-281; Shimamoto, 1994, Current Opinion Biotechnology 5: 158-162;
Vasil et al., 1992, Bio/Technology 10: 667-674). An alternative
method for transformation of monocots is based on protoplast
transformation as described by Omirulleh et al., 1993, Plant
Molecular Biology 21: 415-428.
[0134] Following transformation, the transformants having
incorporated therein the expression construct are selected and
regenerated into whole plants according to methods well-known in
the art.
[0135] The present invention also relates to methods for producing
a polypeptide of the present invention comprising (a) cultivating a
transgenic plant or a plant cell comprising a nucleic acid sequence
encoding a polypeptide having haloperoxidase activity of the
present invention under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
[0136] Compositions
[0137] In a still further aspect, the present invention relates to
compositions comprising a polypeptide of the present invention.
Preferably, the compositions are enriched in a polypeptide of the
present invention. In the present context, the term "enriched"
indicates that the haloperoxidase activity of the composition has
been increased, e.g., with an enrichment factor of 1.1.
[0138] The composition may comprise a polypeptide of the invention
as the major enzymatic component, e.g., a mono-component
composition. Alternatively, the composition may comprise multiple
enzymatic activities, such as an aminopeptidase, amylase,
carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,
cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,
esterase, alpha-galactosidase, beta-galactosidase, glucoamylase,
alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase,
laccase, lipase, mannosidase, oxidase, pectinolytic enzyme,
peptidoglutaminase, peroxidase, phytase, polyphenoloxidase,
proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.
The additional enzyme(s) may be producible by means of a
microorganism belonging to the genus Aspergillus, preferably
Aspergillus aculeatus, Aspergillus awamori, Aspergillus niger, or
Aspergillus oryzae, or Trichoderma, Humicola, preferably Humicola
insolens, or Fusarium, preferably Fusarium bactridioides, Fusarium
cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium
graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium
negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum,
Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum,
Fusarium toruloseum, Fusarium trichothecioides, or Fusarium
venenatum.
[0139] The polypeptide compositions may be prepared in accordance
with methods known in the art and may be in the form of a liquid or
a dry composition. For instance, the polypeptide composition may be
in the form of a granulate or a microgranulate. The polypeptide to
be included in the composition may be stabilized in accordance with
methods known in the art.
[0140] Examples are given below of preferred uses of the
polypeptide compositions of the invention. The dosage of the
polypeptide composition of the invention and other conditions under
which the composition is used may be determined on the basis of
methods known in the art.
[0141] Detergent Composition
[0142] The haloperoxidase of the invention may be added to and thus
become a component of a detergent composition.
[0143] The detergent composition of the invention may for example
be formulated as a hand or machine laundry detergent composition
including a laundry additive composition suitable for pre-treatment
of stained fabrics and a rinse added fabric softener composition,
or be formulated as a detergent composition for use in general
household hard surface cleaning operations, or be formulated for
hand or machine dishwashing operations.
[0144] In a specific aspect, the invention provides a detergent
additive comprising the haloperoxidase of the invention. The
detergent additive as well as the detergent composition may
comprise one or more other enzymes such as a protease, a lipase, a
cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a
mannanase, an arabinase, a galactanase, a xylanase, an oxidase,
e.g., a laccase, and/or a peroxidase.
[0145] In general the properties of the chosen enzyme(s) should be
compatible with the selected detergent, (i.e. pH-optimum,
compatibility with other enzymatic and non-enzymatic ingredients,
etc.), and the enzyme(s) should be present in effective amounts.
Proteases: Suitable proteases include those of animal, vegetable or
microbial origin. Microbial origin is preferred. Chemically
modified or protein engineered mutants are included. The protease
may be a serine protease or a metallo protease, preferably an
alkaline microbial protease or a trypsin-like protease. Examples of
alkaline proteases are subtilisins, especially those derived from
Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin
309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).
Examples of trypsin-like proteases are trypsin (e.g. of porcine or
bovine origin) and the Fusarium protease described in WO 89/06270
and WO 94/25583.
[0146] Examples of useful proteases are the variants described in
WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially
the variants with substitutions in one or more of the following
positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170,
194, 206, 218, 222, 224, 235 and 274.
[0147] Preferred commercially available protease enzymes include
Alcalase.TM., Savinase.TM., Primase.TM., Everlase.TM.,
Esperase.TM., and Kannase.TM. (Novozymes A/S), Maxatase.TM.,
Maxacal.TM., Maxapem.TM., Properase.TM., Purafect.TM., Purafect
OxP.TM., FN2.sup..TM., and FN3.TM. (Genencor International
Inc.).
[0148] Lipases: Suitable lipases include those of bacterial or
fungal origin. Chemically modified or protein engineered mutants
are included. Examples of useful lipases include lipases from
Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T.
lanuginosus) as described in EP 258 068 and EP 305 216 or from H.
insolens as described in WO 96/13580, a Pseudomonas lipase, e.g.
from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P.
cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens,
Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P.
wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B.
subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta,
1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus
(WO 91/16422).
[0149] Other examples are lipase variants such as those described
in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381,
WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO
97/04079 and WO 97/07202.
[0150] Preferred commercially available lipase enzymes include
Lipolase.TM. and Lipolase Ultra.TM. (Novozymes A/S).
[0151] Amylases: Suitable amylases (.alpha. and/or .beta.) include
those of bacterial or fungal origin. Chemically modified or protein
engineered mutants are included. Amylases include, for example,
.alpha.-amylases obtained from Bacillus, e.g. a special strain of
B. licheniformis, described in more detail in GB 1,296,839.
[0152] Examples of useful amylases are the variants described in WO
94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the
variants with substitutions in one or more of the following
positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188,
190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.
[0153] Commercially available amylases are Duramyl.TM.,
Termamyl.TM., FungaMyl.TM. and BAN.TM. (Novozymes A/S),
Rapidase.TM. and Purastar.TM. (from Genencor International
Inc.).
[0154] Cellulases: Suitable cellulases include those of bacterial
or fungal origin. Chemically modified or protein engineered mutants
are included. Suitable cellulases include cellulases from the
genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia,
Acremonium, e.g. the fungal cellulases produced from Humicola
insolens, Myceliophthora thermophila and Fusarium oxysporum
disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178,
5,776,757 and WO 89/09259.
[0155] Especially suitable cellulases are the alkaline or neutral
cellulases having colour care benefits. Examples of such cellulases
are cellulases described in EP 0 495 257, EP 0 531 372, WO
96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase
variants such as those described in WO 94/07998, EP 0 531 315, U.S.
Pat. Nos. 5,457,046, 5,686,593, 5,763,254, WO 95/24471, WO 98/12307
and PCT/DK98/00299.
[0156] Commercially available cellulases include Celluzyme.TM., and
Carezyme.TM.(Novozymes A/S), Clazinase.TM., and Puradax HA.TM.
(Genencor International Inc.), and KAC-500(B).TM. (Kao
Corporation).
[0157] Peroxidases/Oxidases: Suitable peroxidases/oxidases include
those of plant, bacterial or fungal origin. Chemically modified or
protein engineered mutants are included. Examples of useful
peroxidases include peroxidases from Coprinus, e.g. from C.
cinereus, and variants thereof as those described in WO 93/24618,
WO 95/10602, and WO 98/15257.
[0158] Commercially available peroxidases include Guardzyme.TM.
(Novozymes A/S).
[0159] The detergent enzyme(s) may be included in a detergent
composition by adding separate additives containing one or more
enzymes, or by adding a combined additive comprising all of these
enzymes. A detergent additive of the invention, i.e. a separate
additive or a combined additive, can be formulated e.g. as a
granulate, a liquid, a slurry, etc. Preferred detergent additive
formulations are granulates, in particular non-dusting granulates,
liquids, in particular stabilized liquids, or slurries.
[0160] Non-dusting granulates may be produced, e.g., as disclosed
in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be
coated by methods known in the art. Examples of waxy coating
materials are poly(ethylene oxide) products (polyethyleneglycol,
PEG) with mean molar weights of 1000 to 20000; ethoxylated
nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated
fatty alcohols in which the alcohol contains from 12 to 20 carbon
atoms and in which there are 15 to 80 ethylene oxide units; fatty
alcohols; fatty acids; and mono- and di- and triglycerides of fatty
acids. Examples of film-forming coating materials suitable for
application by fluid bed techniques are given in GB 1483591. Liquid
enzyme preparations may, for instance, be stabilized by adding a
polyol such as propylene glycol, a sugar or sugar alcohol, lactic
acid or boric acid according to established methods. Protected
enzymes may be prepared according to the method disclosed in EP
238,216.
[0161] The detergent composition of the invention may be in any
convenient form, e.g., a bar, a tablet, a powder, a granule, a
paste or a liquid. A liquid detergent may be aqueous, typically
containing up to 70% water and 0-30% organic solvent, or
non-aqueous.
[0162] The detergent composition comprises one or more surfactants,
which may be non-ionic including semi-polar and/or anionic and/or
cationic and/or zwitterionic. The surfactants are typically present
at a level of from 0.1% to 60% by weight.
[0163] When included therein the detergent will usually contain
from about 1% to about 40% of an anionic surfactant such as linear
alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty
alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,
alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid
or soap.
[0164] When included therein the detergent will usually contain
from about 0.2% to about 40% of a non-ionic surfactant such as
alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside,
alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide,
fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or
N-acyl N-alkyl derivatives of glucosamine ("glucamides").
[0165] The detergent may contain 0-65% of a detergent builder or
complexing agent such as zeolite, diphosphate, triphosphate,
phosphonate, carbonate, citrate, nitrilotriacetic acid,
ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic
acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered
silicates (e.g. SKS-6 from Hoechst).
[0166] The detergent may comprise one or more polymers. Examples
are carboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene
glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide),
poly(vinylimidazole), polycarboxylates such as polyacrylates,
maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid
copolymers.
[0167] The detergent may contain a bleaching system, which may
comprise a H.sub.2O.sub.2 source such as perborate or percarbonate
which may be combined with a peracid-forming bleach activator such
as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.
Alternatively, the bleaching system may comprise peroxyacids of
e.g. the amide, imide, or sulfone type.
[0168] The enzyme(s) of the detergent composition of the invention
may be stabilized using conventional stabilizing agents, e.g., a
polyol such as propylene glycol or glycerol, a sugar or sugar
alcohol, lactic acid, boric acid, or a boric acid derivative, e.g.,
an aromatic borate ester, or a phenyl boronic acid derivative such
as 4-formylphenyl boronic acid, and the composition may be
formulated as described in e.g. WO 92/19709 and WO 92/19708.
[0169] The detergent may also contain other conventional detergent
ingredients such as e.g. fabric conditioners including clays, foam
boosters, suds suppressors, anti-corrosion agents, soil-suspending
agents, anti-soil redeposition agents, dyes, bactericides, optical
brighteners, hydrotropes, tarnish inhibitors, or perfumes.
[0170] It is at present contemplated that in the detergent
compositions any enzyme, in particular the haloperoxidase of the
invention, may be added in an amount corresponding to 0.01-100 mg
of enzyme protein per liter of wash liqour, preferably 0.05-5 mg of
enzyme protein per liter of wash liquor, in particular 0.1-1 mg of
enzyme protein per liter of wash liquor.
[0171] The haloperoxidase of the invention may additionally be
incorporated in the detergent formulations disclosed in WO
97/07202, which is hereby incorporated as reference.
[0172] Uses
[0173] The present invention is also directed to methods for using
the polypeptides having haloperoxidase activity.
[0174] The present invention is further directed to methods of
oxidizing a halide ion to the corresponding hypohalous acid,
comprising reacting the halide ion and a source of hydrogen
peroxide in the presence of a haloperoxidase of the invention. The
present invention also relates to methods of halogenating a
compound comprising reacting the compound, a halide ion and a
source of hydrogen peroxide in the presence of a haloperoxidase of
the invention.
[0175] The present invention also relates to methods for killing or
inhibiting the growth of microbial cells, comprising contacting the
cells with a haloperoxidase of the invention, a source of hydrogen
peroxide, and a source of halide or thiocyanate in an aqueous
solution.
[0176] The source of hydrogen peroxide can be hydrogen peroxide
itself or a hydrogen peroxide precursor, such as, a percarbonate,
perborate, peroxycarboxylic acid or a salt thereof. Furthermore,
the source may be a hydrogen peroxide generating enzyme system,
such as an oxidase, e.g., a glucose oxidase, glycerol oxidase or
amino acid oxidase, and its substrate. The hydrogen peroxide source
may be added in a concentration corresponding to a hydrogen
peroxide concentration in the range of from about 0.001 to about 10
mM, preferably about 0.01 to about 1 mM.
[0177] The halide source may be a halide salt, preferably a sodium
or potassium salt, such as sodium chloride, potassium chloride,
sodium bromide, potassium bromide, sodium iodide, or potassium
iodide. The thiocyanate source may be a thiocyanate salt,
preferably a sodium or potassium salt.
[0178] The concentration of the halide source will typically
correspond to 0.001-1000 mM, preferably in the range of from
0.005-500 mM, more preferably in the range of from 0.01-100 mM, and
most preferably in the range of from 0.05-50 mM.
[0179] The haloperoxidases may be used as preservation agents and
disinfection agents such as in water based paints and personal care
products, e.g., toothpaste, mouthwash, skin care creams and
lotions, hair care and body care formulations, solutions for
cleaning contact lenses and dentures. The haloperoxidases also may
be used for cleaning surfaces and cooking utensils in food
processing plants and in any area in which food is prepared or
served. The haloperoxidases also may be used in enzymatic bleaching
applications, e.g., pulp bleaching and stain bleaching (in
detergent compositions).
[0180] The concentration of the haloperoxidase in the methods of
use of the present invention, is preferably in the range of 0.01-50
mg/l, more preferably in the range of 0.1-10 mg/l.
[0181] DNA recombination (shuffling)
[0182] The nucleotide sequence of SEQ ID NO:1 may be used in a DNA
recombination (or shuffling) process. The new polynucleotide
sequences obtained in such a process may encode new polypeptides
having haloperoxidase activity with improved properties, such as
improved stability (storage stability, thermostability), improved
specific activity, improved pH-optimum, and/or improved tolerance
towards specific compounds.
[0183] Shuffling between two or more homologous input
polynucleotides (starting-point polynucleotides) involves
fragmenting the polynucleotides and recombining the fragments, to
obtain output polynucleotides (i.e. polynucleotides that have been
subjected to a shuffling cycle) wherein a number of nucleotide
fragments are exchanged in comparison to the input
polynucleotides.
[0184] DNA recombination or shuffling may be a (partially) random
process in which a library of chimeric genes is generated from two
or more starting genes. A number of known formats can be used to
carry out this shuffling or recombination process.
[0185] The process may involve random fragmentation of parental DNA
followed by reassembly by PCR to new full-length genes, e.g. as
presented in U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721,
6,117,679. In-vitro recombination of genes may be carried out, e.g.
as described in U.S. Pat. No. 6,159,687, W/O 98/41623, U.S. Pat.
Nos.6,159,688, 5,965,408, 6,153,510. The recombination process may
take place in vivo in a living cell, e.g. as described in WO
97/07205 and WO 98/28416.
[0186] The parental DNA may be fragmented by DNA'se I treatment or
by restriction endonuclease digests as descriobed by Kikuchi et al
(2000a, Gene 236:159-167). Shuffling of two parents may be done by
shuffling single stranded parental DNA of the two parents as
described in Kikuchi et al (2000b, Gene 243:133-137).
[0187] A particular method of shuffling is to follow the methods
described in Crameri et al, 1998, Nature, 391: 288-291 and Ness et
al. Nature Biotechnology 17: 893-896. Another format would be the
methods described in U.S. Pat. No. 6,159,687: Examples 1 and 2.
[0188] The present invention is further described by the following
examples, which should not be construed as limiting the scope of
the invention.
EXAMPLES
[0189] Chemicals used as buffers and substrates were commercial
products of at least reagent grade.
[0190] Haloperoxidase assays
[0191] Microtiter assays are performed by mixing 100 .mu.l of
haloperoxidase sample (about 0.2 .mu.g/ml) and 100 .mu.l assay
buffer (0.3 M sodium phosphate; pH 7; 1.25 mM Na.sub.3VO.sub.4; 50
mM KBr; 0.008% phenol red). Reactions were initiated by adding 10
.mu.l of 0.3% H.sub.2O.sub.2, and the absorption at 595 nm was
measured spectrophotometrically as a function of time in a
Molecular Devices Kinetic Microplate reader.
[0192] Assays using monochlorodimedone (Sigma M4632,
.epsilon.=20000M.sup.-1cm.sup.-1 at 290 nm) as a substrate are
performed as described below. The decrease in absorption at 290 nm
is measured as a function of time. Assays are performed in 0.1 M
sodium phosphate or 0.1 M sodium acetate, 50 .mu.M
monochlorodimedone, 10 mM KBr/KCl, and 1 mM H.sub.2O.sub.2 using a
haloperoxidase concentration of about 1 .mu.g/ml. One HU is defined
as 1 micromol of monochlorodimedone chlorinated or brominated per
minute at pH 5 and 30.degree. C.
Example 1
[0193] Transformation and fermentation of Aspergillus oryzae
[0194] Protoplast preparation and transformation in Aspergillus
oryzae of the nucleic acid sequence encoding the Geniculosporium
sp. haloperoxidase contained in the plasmid contained in E. coli
DH10B, deposited as DSM 13442, was done essentially as described by
Christensen et al. (1988), Biotechnology, 6:1419-1422.
Transformants were plated on AMDS agar plates selecting for the
ability to grow on acetamide as sole nitrogen source.
[0195] A. oryzae transformants were spore purified twice and
inoculated into 100 .mu.l YP growth medium supplemented with
maltose (3%), 1 mM Na.sub.3VO.sub.4, and 0.4% Urea. Cultures were
grown at 34.degree. C. for 5 days after which they were assayed for
haloperoxidase activity. The best haloperoxidase producer from the
transformation was inoculated into 8.times.125 ml baffled flasks
containing YP growth medium with maltose (3%), 1 mM
Na.sub.3VO.sub.4, and 0.4% Urea and grown for 7 days at 34.degree.
C., with shaking at 200 rpm.
Example 2
[0196] Purification of Geniculosporium sp. haloperoxidase
[0197] Glucanex.TM. (available from Novozymes A/S) treated
fermentation broth was centrifuged and the supernatant was filtered
through a Seitz EKS filter plate (Seitz-Filter-Werke GmbH, Germany)
and concentrated and washed by ultrafiltration on a Filtron
Minisette.TM. system (Filtron Technology Corporation,
Massachusetts, USA) with an Omega type membrane (Mw cut-off of 10
kDa) to a final volume of 300 ml and a conductance less than 2
mS.
[0198] The crude enzyme preparation was filtered on a glass filter,
added 6 ml of a 10 mM sodium orthovanadate, and applied onto an
anion exchange column (Pharmacia 26/10 with Q-Sepharose High
Performance) equilibrated with 50 mM Tris/HCl pH 7. The column was
washed with equilibration buffer and eluted with a 0-1M sodium
chloride gradient (over 10 column volumes) in the same buffer using
a flow of 10 ml/minute. Fractions of 10 ml were collected and
tested for haloperoxidase activity.
[0199] Fractions showing haloperoxidase activity were pooled,
concentrated by ultrafiltration on an Amicon cell (membrane Mw
cut-off of 10 kDa) to a final volume of 6-7 ml. Two times 2 ml of
the concentrated sample was applied onto a gel filtration column
(Pharmacia HiLoad 16/60, Su-perdex 200 High Performance)
equilibrated with 50 mM sodium acetate, 100 mM NaCl pH 5.5. The
column was eluted with a flow of 1 ml/minute and fractions of 1 ml
were collected. Fractions from both runs showing HPO activity were
pooled giving 22 ml with A280=10.766. The pooled fractions
contained rather pure haloperoxidase showing only one band on
SDS-PAGE with a Mr close to 65 kDa.
Example 3
[0200] Thermal stability of recombinant Geniculosporium sp.
haloperoxidase
[0201] The thermal stability of Geniculosporium sp. haloperoxidase
was determined by the following procedure:
[0202] The enzyme was diluted to an absorbance at 280 nm of approx.
0.1 in a 0.3 M Tris/HCI buffer pH 7. 0.5 ml portions of the
dilution was incubated at 30, 40, 50, 60, 70, and 80.degree. C.,
respectively, for 15 minutes and then placed on ice-water. A
reference sample of the diluted enzyme was kept at 4.degree. C.
Activity of the samples was measured according to the phenol red
assay in microwell plate using bromide as substrate, and residual
activity was calculated relatively to the reference (stored at
4.degree. C.).
[0203] Conclusion: The residual activity of Geniculosporium sp.
haloperoxidase is at least 60% after 15 minutes incubation at
70.degree. C.
1 TABLE 1 Temperature Residual activity (.degree. C.) (%) 4 100 30
103.3 40 100.2 50 92.8 60 84.5 70 61.9 80 14.6
Example 4
[0204] Antibacterial activity of Geniculosporium sp. haloperoxidase
against Escherichia coli
[0205] The antibacterial activity of the Geniculosporium sp.
haloperoxidase, available from enzymes A/S, DK-2880 Bagsvaerd,
Denmark, was tested with bromide as enhancing agent.
[0206] The antibacterial activity of haloperoxidase was tested in
HEPES-buffer (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic
acid])(pH 7.0) against Escherichia coli with potassium bromide as
electron donor, and hydrogen peroxide was lectron acceptor. The
cells (approximately 10.sup.6 CFU/ml) were incubated with enzyme
for 15 min at 40.degree. C.
[0207] The bactericidal activity was determined by incubation in a
Malthus Flexi M2060 instrument (available from Malthus Instruments
Limited, England). The detection times measured by the Malthus
instrument were converted to CFU/ml (colony forming units pr. ml)by
a calibration curve. Direct measurements were used when enumerating
total survival cells. By the direct measurements, the cell
metabolism was determined by conductance measurements in the growth
substrate. When the conductance change is measurable by the Malthus
instrument, a detection time (dt) will be recorded. The dt's were
converted to colony counts by use of a calibration curve relating
CFU/ml to dt.
2 TABLE 2 KBr Enzyme H.sub.2O.sub.2 Bactericidal activity (mM)
(mg/L) (mM) (log CFU/ml) (doublets) 0 0 0 -0.1/0.1 8 0 0 0.3/0.4 0
0 1 0.2/0.8 8 0 1 0.6/0.5 0 1 0 0.4/0.4 8 1 1 6.4*/6.4*
*corresponds to a total kill of the test organism
[0208] Bactericidal activity is shown in the table as log.sub.10
reduction in the number of living cells (colony forming units),
thus a bactericidal activity of 6 correspond to a kill of 10.sup.6
CFU/ml. A significant bactericidal activity was obtained with the
Geniculosporium sp. haloperoxidase, and no significant bactericidal
activity was obtained with any of the controls.
DEPOSIT OF BIOLOGICAL MATERIAL
[0209] An E. coli DH10B clone containing a haloperoxidase gene from
Geniculosporium sp. (SEQ ID NO:1) inserted into a pUC19 derived
plasmid has been deposited under the terms of the Budapest Treaty
with the Deutsche Sammiung von Mikroorganismen und Zellkulturen
GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig, Germany, and
given the following accession number:
3 Deposit Accession Number Date of Deposit NN049533 DSM 13442 Apr.
12, 2000
[0210] The deposit was made by Novo Nordisk A/S and was later
assigned to Novozymes A/S. The strain has been deposited under
conditions that assure that access to the culture will be available
during the pendency of this patent application to one determined by
the Commissioner of Patents and Trademarks to be entitled thereto
under 37 C.F.R. .sctn.1.14 and 35 U.S.C. .sctn.122. The deposit
represents a substantially pure culture of the deposited strain.
The deposit is available as required by foreign patent laws in
countries wherein counterparts of the subject application, or its
progeny are filed. However, it should be understood that the
availability of a deposit does not constitute a license to practice
the subject invention in derogation of patent rights granted by
governmental action.
[0211] The invention described and claimed herein is not to be
limited in scope by the specific embodiments herein disclosed,
since these embodiments are intended as illustrations of several
aspects of the invention. Any equivalent embodiments are intended
to be within the scope of this invention. Indeed, various
modifications of the invention in addition to those shown and
described herein will become apparent to those skilled in the art
from the foregoing description. Such modifications are also
intended to fall within the scope of the appended claims. In the
case of conflict, the present disclosure including definitions will
control.
[0212] Various references are cited herein, the disclosures of
which are incorporated by reference in their entireties.
Sequence CWU 1
1
2 1 1842 DNA Geniculosporium sp. CDS (1)..(1842) 1 atg gca aca ttc
acc cct atc ccg ctt cct caa att gat gaa ccg gca 48 Met Ala Thr Phe
Thr Pro Ile Pro Leu Pro Gln Ile Asp Glu Pro Ala 1 5 10 15 gag tac
aac acg aac tac gtt ctg tat tgg cac cat gtc ggc ttg gag 96 Glu Tyr
Asn Thr Asn Tyr Val Leu Tyr Trp His His Val Gly Leu Glu 20 25 30
ctt aac cgc gtc aca cat acc gtc gga ggc ccg cag act ggc cca cca 144
Leu Asn Arg Val Thr His Thr Val Gly Gly Pro Gln Thr Gly Pro Pro 35
40 45 atc tct gct aga gct ctg ggc atg ctc cag ttg gct gta cat gat
gcc 192 Ile Ser Ala Arg Ala Leu Gly Met Leu Gln Leu Ala Val His Asp
Ala 50 55 60 tac ttc gcc atc cat ccc tct tct agc ttc ctg acc ttt
ttg aca tct 240 Tyr Phe Ala Ile His Pro Ser Ser Ser Phe Leu Thr Phe
Leu Thr Ser 65 70 75 80 ggc gcc gac aac cct gcc tac gct ctg ccc gag
ttg agc ggc gcg gac 288 Gly Ala Asp Asn Pro Ala Tyr Ala Leu Pro Glu
Leu Ser Gly Ala Asp 85 90 95 gat gcc cgc cag gcg gta gct ggt gca
tct gtt act atg ctg tct atg 336 Asp Ala Arg Gln Ala Val Ala Gly Ala
Ser Val Thr Met Leu Ser Met 100 105 110 ctt tac atg aag ccc cct acc
aac ccc aac ccc aat cct ggc gcc acc 384 Leu Tyr Met Lys Pro Pro Thr
Asn Pro Asn Pro Asn Pro Gly Ala Thr 115 120 125 att tcc gac aac gcc
tat gca cag ctt cag tat gtc att gac aaa tca 432 Ile Ser Asp Asn Ala
Tyr Ala Gln Leu Gln Tyr Val Ile Asp Lys Ser 130 135 140 gta acc gat
gca ccc ggt ggt gta gat gca gcg tcc agc agt ttc aac 480 Val Thr Asp
Ala Pro Gly Gly Val Asp Ala Ala Ser Ser Ser Phe Asn 145 150 155 160
ttc gga aag gca gta gct act gtc ttc ttc aac cta ctc ttc cac gcc 528
Phe Gly Lys Ala Val Ala Thr Val Phe Phe Asn Leu Leu Phe His Ala 165
170 175 ccg ggt gcc tca caa gct ggc tat cac cct aca ccc ggc cca tac
aag 576 Pro Gly Ala Ser Gln Ala Gly Tyr His Pro Thr Pro Gly Pro Tyr
Lys 180 185 190 ttc gac gat gag ccc act cac cct gtc gtc ctt gtt ccc
gtt gac gca 624 Phe Asp Asp Glu Pro Thr His Pro Val Val Leu Val Pro
Val Asp Ala 195 200 205 aac aac ccg gat ggt ccc aag cgg cct ttc cgc
cag tat cac ggc ccg 672 Asn Asn Pro Asp Gly Pro Lys Arg Pro Phe Arg
Gln Tyr His Gly Pro 210 215 220 ttc tat ggc aag act gct aag cgt ttt
gct aca cag act gag cat atg 720 Phe Tyr Gly Lys Thr Ala Lys Arg Phe
Ala Thr Gln Thr Glu His Met 225 230 235 240 att gct gac ccg cca gcc
att cgt tct gcc gtt ggt gag caa gct gaa 768 Ile Ala Asp Pro Pro Ala
Ile Arg Ser Ala Val Gly Glu Gln Ala Glu 245 250 255 tac gat gat agt
att cgt caa atc att gcc atg ggt gga gct acc ggc 816 Tyr Asp Asp Ser
Ile Arg Gln Ile Ile Ala Met Gly Gly Ala Thr Gly 260 265 270 ctc aac
tcc acc aag cgc agc cct ttt cag aca act caa ggc atg ttc 864 Leu Asn
Ser Thr Lys Arg Ser Pro Phe Gln Thr Thr Gln Gly Met Phe 275 280 285
tgg gcc tac gat ggc tcc aac ttg gtc ggc aca cca ccc aga ttt tac 912
Trp Ala Tyr Asp Gly Ser Asn Leu Val Gly Thr Pro Pro Arg Phe Tyr 290
295 300 aac cag att gtc cgc cgc atc gca gtg acg tac aag aag gag gaa
gac 960 Asn Gln Ile Val Arg Arg Ile Ala Val Thr Tyr Lys Lys Glu Glu
Asp 305 310 315 320 ttg act aac agc gaa gtc aac aac gca gac ttt gtc
cgt ctc ctt gct 1008 Leu Thr Asn Ser Glu Val Asn Asn Ala Asp Phe
Val Arg Leu Leu Ala 325 330 335 ctg gtc aac gta gcc tgt gcc gat gca
gga atc ttc tcc tgg aaa gag 1056 Leu Val Asn Val Ala Cys Ala Asp
Ala Gly Ile Phe Ser Trp Lys Glu 340 345 350 aag tgg gaa ttt gaa ttc
tgg cgc cca ctc tct ggt gtt cgt gac gac 1104 Lys Trp Glu Phe Glu
Phe Trp Arg Pro Leu Ser Gly Val Arg Asp Asp 355 360 365 aac ttc cgc
gac cca aat cgc cca gat cgt ggc gac cct ttc tgg ctt 1152 Asn Phe
Arg Asp Pro Asn Arg Pro Asp Arg Gly Asp Pro Phe Trp Leu 370 375 380
act ctc ggc gcc cca gcc aca aac aca aac gac att cct ttc aaa ccc
1200 Thr Leu Gly Ala Pro Ala Thr Asn Thr Asn Asp Ile Pro Phe Lys
Pro 385 390 395 400 ccc ttc ccc gct tac ccc tct ggt cac gcc aca ttc
ggt ggc gcc gtc 1248 Pro Phe Pro Ala Tyr Pro Ser Gly His Ala Thr
Phe Gly Gly Ala Val 405 410 415 ttc cag atg gtc cgc cgc tac tac aac
ggg cga gtt gga aac tgg aaa 1296 Phe Gln Met Val Arg Arg Tyr Tyr
Asn Gly Arg Val Gly Asn Trp Lys 420 425 430 gac gac gaa gtg gac aac
atc gcc atc gat atg atg gta tcc gag gag 1344 Asp Asp Glu Val Asp
Asn Ile Ala Ile Asp Met Met Val Ser Glu Glu 435 440 445 ctc aac ggg
ttg agc cgt gat ctc cgc caa ccc tac gac ccc aaa gcg 1392 Leu Asn
Gly Leu Ser Arg Asp Leu Arg Gln Pro Tyr Asp Pro Lys Ala 450 455 460
ccc att acc gat cag cca ggt atc gtg cgc aca cga gtt cca cgc cac
1440 Pro Ile Thr Asp Gln Pro Gly Ile Val Arg Thr Arg Val Pro Arg
His 465 470 475 480 ttc tct tcc gtc tgg gag atg atg ttc gag aac gca
atc tcg cgt atc 1488 Phe Ser Ser Val Trp Glu Met Met Phe Glu Asn
Ala Ile Ser Arg Ile 485 490 495 ttt ctc ggc gtc cac tgg cgc ttc gat
gct gca gcc gcc aag gat att 1536 Phe Leu Gly Val His Trp Arg Phe
Asp Ala Ala Ala Ala Lys Asp Ile 500 505 510 ttg atc ccc acg acg aca
aag gat gtc tac gct gta gac aac aac ggc 1584 Leu Ile Pro Thr Thr
Thr Lys Asp Val Tyr Ala Val Asp Asn Asn Gly 515 520 525 gct tcc ttg
ttc caa aac gtc gag gat att cgt tat acg act atg ggt 1632 Ala Ser
Leu Phe Gln Asn Val Glu Asp Ile Arg Tyr Thr Thr Met Gly 530 535 540
act agg gag ggt cac gat ggg ctt ttg ccg att ggt ggt gtg ccg ctt
1680 Thr Arg Glu Gly His Asp Gly Leu Leu Pro Ile Gly Gly Val Pro
Leu 545 550 555 560 ggt att ggg att gcg aat gag atc ttt gat aca ggt
ctc aag cct acc 1728 Gly Ile Gly Ile Ala Asn Glu Ile Phe Asp Thr
Gly Leu Lys Pro Thr 565 570 575 cca ccg gag aaa cag cca gtg ccg ccg
cct cca ttc aac cag agc gga 1776 Pro Pro Glu Lys Gln Pro Val Pro
Pro Pro Pro Phe Asn Gln Ser Gly 580 585 590 cct acg aag gag atg ttg
gag gaa gcg gga agt gag gag cag gtc cct 1824 Pro Thr Lys Glu Met
Leu Glu Glu Ala Gly Ser Glu Glu Gln Val Pro 595 600 605 atg atg gac
gtt gcg ccc 1842 Met Met Asp Val Ala Pro 610 2 614 PRT
Geniculosporium sp. 2 Met Ala Thr Phe Thr Pro Ile Pro Leu Pro Gln
Ile Asp Glu Pro Ala 1 5 10 15 Glu Tyr Asn Thr Asn Tyr Val Leu Tyr
Trp His His Val Gly Leu Glu 20 25 30 Leu Asn Arg Val Thr His Thr
Val Gly Gly Pro Gln Thr Gly Pro Pro 35 40 45 Ile Ser Ala Arg Ala
Leu Gly Met Leu Gln Leu Ala Val His Asp Ala 50 55 60 Tyr Phe Ala
Ile His Pro Ser Ser Ser Phe Leu Thr Phe Leu Thr Ser 65 70 75 80 Gly
Ala Asp Asn Pro Ala Tyr Ala Leu Pro Glu Leu Ser Gly Ala Asp 85 90
95 Asp Ala Arg Gln Ala Val Ala Gly Ala Ser Val Thr Met Leu Ser Met
100 105 110 Leu Tyr Met Lys Pro Pro Thr Asn Pro Asn Pro Asn Pro Gly
Ala Thr 115 120 125 Ile Ser Asp Asn Ala Tyr Ala Gln Leu Gln Tyr Val
Ile Asp Lys Ser 130 135 140 Val Thr Asp Ala Pro Gly Gly Val Asp Ala
Ala Ser Ser Ser Phe Asn 145 150 155 160 Phe Gly Lys Ala Val Ala Thr
Val Phe Phe Asn Leu Leu Phe His Ala 165 170 175 Pro Gly Ala Ser Gln
Ala Gly Tyr His Pro Thr Pro Gly Pro Tyr Lys 180 185 190 Phe Asp Asp
Glu Pro Thr His Pro Val Val Leu Val Pro Val Asp Ala 195 200 205 Asn
Asn Pro Asp Gly Pro Lys Arg Pro Phe Arg Gln Tyr His Gly Pro 210 215
220 Phe Tyr Gly Lys Thr Ala Lys Arg Phe Ala Thr Gln Thr Glu His Met
225 230 235 240 Ile Ala Asp Pro Pro Ala Ile Arg Ser Ala Val Gly Glu
Gln Ala Glu 245 250 255 Tyr Asp Asp Ser Ile Arg Gln Ile Ile Ala Met
Gly Gly Ala Thr Gly 260 265 270 Leu Asn Ser Thr Lys Arg Ser Pro Phe
Gln Thr Thr Gln Gly Met Phe 275 280 285 Trp Ala Tyr Asp Gly Ser Asn
Leu Val 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 Thr
Asn Ser Glu Val Asn Asn Ala Asp Phe Val Arg Leu Leu Ala 325 330 335
Leu Val Asn Val Ala Cys Ala 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 Asn Phe Arg Asp Pro Asn Arg Pro Asp Arg Gly Asp Pro
Phe Trp Leu 370 375 380 Thr Leu Gly Ala Pro Ala Thr Asn Thr Asn Asp
Ile Pro Phe Lys Pro 385 390 395 400 Pro Phe Pro Ala Tyr Pro Ser Gly
His Ala Thr Phe Gly Gly Ala Val 405 410 415 Phe Gln Met Val Arg Arg
Tyr Tyr Asn Gly Arg Val Gly Asn Trp Lys 420 425 430 Asp Asp Glu Val
Asp Asn Ile Ala Ile Asp Met Met Val Ser Glu Glu 435 440 445 Leu Asn
Gly Leu Ser Arg Asp Leu Arg Gln Pro Tyr Asp Pro Lys Ala 450 455 460
Pro Ile Thr Asp Gln Pro Gly Ile Val Arg Thr Arg Val Pro Arg His 465
470 475 480 Phe Ser Ser Val Trp Glu Met Met Phe Glu Asn Ala Ile Ser
Arg Ile 485 490 495 Phe Leu Gly Val His Trp Arg Phe Asp Ala Ala Ala
Ala Lys Asp Ile 500 505 510 Leu Ile Pro Thr Thr Thr Lys Asp Val Tyr
Ala Val Asp Asn Asn Gly 515 520 525 Ala Ser Leu Phe Gln Asn Val Glu
Asp Ile Arg Tyr Thr Thr Met Gly 530 535 540 Thr Arg Glu Gly His Asp
Gly Leu Leu Pro Ile Gly Gly Val Pro Leu 545 550 555 560 Gly Ile Gly
Ile Ala Asn Glu Ile Phe Asp Thr Gly Leu Lys Pro Thr 565 570 575 Pro
Pro Glu Lys Gln Pro Val Pro Pro Pro Pro Phe Asn Gln Ser Gly 580 585
590 Pro Thr Lys Glu Met Leu Glu Glu Ala Gly Ser Glu Glu Gln Val Pro
595 600 605 Met Met Asp Val Ala Pro 610
* * * * *