Note: Descriptions are shown in the official language in which they were submitted.
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Phenolic Acid Esterase and Use Thereof
The present invention relates to an enzyme with phenolic acid
esterase activity, DNA molecule encoding said enzyme as well
as a method for the production and use of said enzyme.
Background
Plant cell walls are divided into two sections, the primary
and secondary cell wall. The primary cell wall is comprised
of three major classes of polysaccharides: cellulose,
hemicellulose and pectin. The secondary cell wall also
contains polysaccharides as well as lignin. Hemicellulose, a
general class of highly branched polysaccharides found in the
plant cell wall, is bound to itself as well as to cellulose
and lignin, and these bonds contribute to the stability and
support of the plant structure.
Hemicelluloses based on a xylose backbone are designated as
xylans. Xylan, which has been shown to exist in a wide
variety of different plants including fruits, vegetables
legumes, cereals, grasses, softwoods and hardwoods, is a
linear (3-(1-4)-D-xylopyranose pol~,nner which can be substituted
with sugar residues, including a-L-arabinose, and c~.-D-
glucuronic acid and/or the 4-0-methyl ether derivative of cc-D-
glucuronic acid. Many xylans are also esterified with
phenolic acid residues, including coumaric acid and ferulic
acid. These phenolic acid residues are present in an ester
linkage to a-L-arabinofuranosyl xylan and can serve to protect
xylan from xylan-degrading enzymes, so-called xylanases, as
well they confer structural stability to the plant cell wall
by forming covalent bonds with the lignin present therein.
In addition, ferulic acid has been shown to exist as a
diferulic acid bridge between different xylan chains,
imparting further structural support for plant cells (Linden,
J.C. et al., American Chemical Society Symposium Series, vol.
566 (1994), 452-467).
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A number of microorganisms are known which are capable of
hydrolysing phenolic acid esters and digesting plant cell
walls through the enzymatic breakdown of plant cell wall
polysaccharides. Some of these microorganisms possess
enzymes) with phenolic acid esterase activity, i.e. coumaric
acid esterase activity or ferulic acid esterase activity or a
combination of these two activities.
For example, Borneman, W.S. et al (Applied and Environmental
Microbiology, vol. 58 (1992), 3762-3766) describe two ferulic
acid esterases (FAE), designated FAE-I and FAE-II
respectively, isolated from the anaerobic fungus
Neocallimastix strain MC-2. FAE-II was reported to be
specific for the substrate (O-{5-O-[(E)-feruloyl]-a.-L-
arabinofuranosyl~-(1-3)-O-~-D-xylopyranosyl-(1-4)-D- _
xylopyranose (FAXX), whereas FAE-I was reported to have both a
FAXX degrading activity as well as a (0-{5-0-[(E)-p-
coumaroyl]-a-L-arabinofuranosyl}-(1-3)-O-(i-D-xylopyranosyl-(1-
4)-D-xylopyranose (PAXX) degrading activity, the maximum ratio
of metabolism of FAXX:PAXX being 3:1. The pH optima of these
two enzymes were shown to be 6.2 and 7.0 respectively when
using FAXX as a substrate.
GB 2 301 103 discloses an FAE obtained from Aspergillus niger
as well as the gene encoding said enzyme . Said enzyme has a
pH optimum of about 5 and a temperature optimum of from about
50 to 60°C when methyl ferulate is used as a substrate.
Other purified enzymes with ferulic acid esterase activity are
known (for example, see McCrae, S.I. et al., Enzyme Microb.
Technol., vol. 16 (1994), 826-834; Faulds, C.B. and
Williamson, G., Microbiology, vol. 140 (1994), 779-787;
Castanares, A. et al., Enzyme Microb. Technol., vol. 14
(1992), 875; and Kroon, P.A. et al., Biotechnol. Appl.
Biochem., vol 23 (1996), 255-262) which have pH optima ranging
from about 5.0 to 6.0 and temperature optima from 30 to 60°C.
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Enzymes with phenolic acid esterase activity can be used in a
number of industrial, agricultural and health applications
which can be carried out at pH values about or above 6.5
and/or at temperatures above 45°C.
Summary of the invention
It is an object of the present invention to provide enzyme
with good phenolic acid esterase activity.
In addition, it is an object of the present invention to
provide a source of an enzyme with phenolic acid esterase
activity which is available in relatively large amounts.
Furthermore, it is an object t:o provide a method for the
production of an enzyme with pher~olic acid esterase activity.
A further object is to provide uses of an enzyme phenolic acid
esterase activity for the preparation of food and feed, for
the processing of paper and pulp as well as for the
bioconversion of ligno-cellulose wastes, for example.
Other objects of the present invention will become apparent
from the following detailed description.
Subject matter of the present invention is an enzyme with
phenolic acid esterase activity, characterized in that said
enzyme has a pH optimum greater than pH 6.5, preferably about
7.0, and a temperature optimum greater than 45°C, preferably
greater than 50°C, and more preferably about 55°C, when
measured in a citric acid/disodium hydrogen orthophosphate
buffer containing 33 ~,M FAXX as a substrate. Preferably, said
enzyme has ferulic acid esterase activity and coumaric acid
esterase activity.
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Subject matter of the present invention is also an enzyme with
phenolic acid esterase activity obtainable from Piromyces Sp.,
for example Piromyces equi, and more preferably from the
Piromyces equi strain deposited under the Budapest Treaty at
the International Mycological Institute (IMI), Bakeham Lane,
Egham, Surrey, UK under the Accession Number 375061.
Preferably, the enzyme of the present invention comprises the
amino acid sequence given in SEQ ID NO:1 or a functional
derivative thereof. A functional derivative of the enzyme of
the present invention is defined as an enzyme having one or
more N-terminal, C-terminal or internal substitution(s),
insertions) and/or deletions) in the amino acid sequence
given in SEQ ID NO:1 which maintains a pH optimum greater than
6.5, preferably about 7.0, and a temperature optimum greater
than 45°C, preferably greater than 50°C, and more preferably
about 55°C, when measured in a citric acid/disodium hydrogen
orthophosphate buffer containing 33 ~,M FAXX as a substrate.
More preferably, the enzyme of the present invention comprises
the amino acid sequence given in SEQ ID N0:3 or a functional
derivative thereof.
In addition, the enzyme of the present invention is preferably
encoded by the DNA sequence given in SEQ ID N0:1 or a
functional derivative thereof. More preferably, the enzyme of
the present invention is encoded by the DNA sequence given in
SEQ ID N0:3 or a functional derivative thereof.
The present invention relates to a phenolic acid esterase with
one or more of the above properties.
Further subject matter of the present invention is a DNA
molecule encoding an enzyme with phenolic acid esterase
activity, characterized in that said DNA molecule comprises a
DNA sequence as given in SEQ ID N0:1 or a functional
derivative or homologue thereof. A functional derivative of
the DNA sequence given in SEQ ID N0:1 is defined as a DNA
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sequence having one or more 5'-, 3'- or internal
substitution(s), insertions) and/or deletions) in the DNA
sequence given in SEQ ID NO:1 which maintains its capability
to encode an enzyme with phenolic acid esterase activity which
has a pH optimum greater than 6.5, preferably about 7.0, and a
temperature optimum greater than 45°C, preferably greater than
50°C, and more preferably about 55°C, when measured in a citric
acid/disodium hydrogen orthophosphate buffer comprising 33 uM
FAXX as a substrate. A functional homologue of the DNA
sequence of the present invention is defined as a DNA sequence
with preferably 75o homology, more preferably 85o homology and
most preferably 95o homology to the DNA sequence given in SEQ
ID NO:1 or SEQ~ID N0:3. More preferably, a DNA molecule
encoding an enzyme according to the present invention
comprises a DNA sequence as given in SEQ ID N0:3 or a
functional derivative or homologue thereof.
In a preferred embodiment, DNA molecules of the present
invention comprise vector sequence capable of replicating said
DNA molecules and/or expressing said enzyme in a procaryotic
or eucaryotic host.
Further subject matter of the present invention is a
transformed procaryotic cell or eucaryotic cell comprising one
or more DNA molecules of the present invention. Preferably
said cells are selected from the group comprising E. coli,
Bacillus sp., such as Bacillus subtilis, Lactobacillus sp.,
and Lactococcus sp., Aspergillus, Trichoderma, Penicillium,
Mucor, Kluyveromyces and Saccharomyces, such as Saccharomyces
cerevisiae.
The enzyme of the present invention may be expressed in
transgenic plants such as maize, soybean and canola/rapeseed.
or in root storage organs of plants, such as potato, carrot
and sugar beet.
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The introduction of an esterase of the present invention
expressed and/or secreted at the appropriate stage, for
example, at harvest, has the advantage that the risk of
weakening the transgenic plant or storage root organ structure
during growth can be reduced.
The methodology for the production of transformed procaryotic
and eucaryotic cells is known in the art. Transgenic fungus,
such as Aspergillus, tranformed yeast, such as Saccharomyces,
and transgenic plants are also known inthe art and can be
produced by the methods taught and discussed in GB 2 301 103,
EP 479 359 and EP 449 375.
Subject matter of the present invention is also a method for
the production of an enzyme or enzyme preparation having
phenolic acid esterase activity, characterized in that said
enzyme is isolated from a naturally occurring organism or
transformed cell or organism capable of expressing the enzyme
according to the invention. Enzyme preparations including, for
example, partially purified preparations obtainable as a cell
or organism extract are also subject matter of the present
invention.
The enzyme preparation of the present invention can comprise
one or more further polysaccharide modifying and/or degrading
enzymes. Said polysaccharide modifying and/or degrading
enzymes) is (are) preferably selected from the group
comprising xylanase, arabinanase, a,-L-arabinofuranosidase,
endoglucanase, a-D-glucuronidase, pectinase, acetyl esterase,
mannanase, acetyl xylan esterase and other glycosyl
hydrolases.
In addition, the enzyme preparation of the present inventio:~
can also include one or more further enzymes selected from the
group comprising amylase, protease, a-galactosidase, phytase
and lipase.
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Use of the enzyme and/or enzyme preparation according to the
invention include the use in a process for releasing phenolic
acids from a substrate comprising phenolic acid moieties.
Said enzyme and/or enzyme preparation according to the
invention can equally find use in the production of animal
feed by improving the digestibility of plant material,
especially forage in which the plant cell walls have a high
phenolic acid content. Furthermore, the enzyme and/or the
enzyme preparation according to the invention can be used in
or with crop plants including but not limited to maize, wheat,
grasses and alfalfa, to improve the digestability for
livestock by pre-modifying the cell wall content. Said enzyme
and/or enzyme preparation according to the invention can also
find used in the preparation of food for human consumption.
Further subject matter of the present invention is also a feed
additive comprising an enzyme or enzyme preparation having
phenoiic acid esterase activity according to the invention and
a feed comprising said feed additive.
The enzyme and/or enzyme preparation according to the
invention can also find use in the paper and pulp industry,
for example, in helping remove lignin from cellulose pulps.
Additionally, used in combination with xylan degrading
enzymes, the enzyme and/or enzyme preparation according to the
invention can contribute to a reduction in the amount of
chlorine required for bleaching by increasing the solubility
and extractability of lignin from pulp.
Furthermore, when combined witrF xylanases and/or cellulases,
the enzyme and/or enzyme preparation according to the
invention can be used for the bioconversion of plant material
or ligno-cellulose wastes to sugars, for example, for chemical
or fuel production, and/or in the production of phenolic
acids.
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Brief description of the drawings
Figure 1: pH profile of the phenolic acid esterase of the
present invention measured using FAXX as a
substrate.
Figure 2: Temperature profile of the phenolic acid esterase of
the present invention measured using FAXX as a
substrate.
Detailed description of the invention
The following Examples are intended to more closely illustrate
the present invention without limiting the subject matter of
the invention to said Examples.
Example 1
Piromyces equi isolated from horse cecum (Orpin, C.G., J. Gen.
Microbiol., vol. 123 (1981), 287-296) and as described by E.A.
Munn in Anaerobic Fungi, Biology, Ecology and Function, D.O.
Mountfort and C.G. Orpin Eds., Marcel Dekker, Inc., New York,
1994, 47-105, and deposited under the Budapest Treaty at the
International Mycological Institute (IMI) under the Accession
Number 375061 was cultured under anaerobic conditions at a
temperature of 39°C in a rumen fluid-containing medium
(Kemp,P., Lander, D.J. and Orpin, C.G., J. Gen. Microbiol.,
vol. 130 (1984), 27-37) with 0.100 soluble xylan and 0.5%
Sigmacell (Sigma Chemical Co., Poole, Dorset, England) as
carbon sources. Total RNA was extracted from fungus grown
under the above conditions, poly(A)+ RNA was selected by
oligo(dT) chromatography, and double-stranded cDNA was
synthesized from the selected RNA, cloned into ~,ZAPII using a
ZAP-cDNA synthesis kit and packaged in vitro according to the
instructions of the manufacturer (Stratagene, La Jolla,
California, USA) (Xue, G-P. et al., J Gen. Microbiol., vol.
138 (1992), 1413-1420 and Ali, B.R.S. et al., FEMS Microbiol.
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Lett., vol. 125 (1995), 15-22). Recombinant phage were grown
by plating on lawns of E . coli XL1-Blue in soft agar overlays
and screened using an antibody raised against a fungal
cellulase/hemicellulase complex purified according to
Ali,B.R.S. et al., FEMS Microbiol_. Lett., vol. 125 (1995), 15-
22). Antibody screening of phage plaques with rabbit anti-
complex antibody as the primary antibody was carried out
essentially as described in thE: instruction manual provided
with the picoBlueT''t immunoscreeni.ng kit (Stratagene) , with the
following modifications: isopropyl-~i-D-thiogalactopyranoside
(IPTG: 0.33 mM) was added directly to the soft agar overlays
containing recombinant a.ZAPII and host bacteria (E. coli XL1-
Blue); plaques were lifted onto Hybond-C filters (Amersham);
blocking solution contained dried milk powder (4o w/v) in
place of BSA; anti-rabbit IgG (whole molecule) conjugated to
horseradish peroxidase (Sigma Chemical Co.) was used as
secondary antibody; colour development solution comprised
3,3'-diaminobenzidine (0.5 mg/ml) in 50 mM Tris-HC1 buffer, pH
7.4, containing hydrogen peroxide (0.5 ~.l/ml. Esterase
production was verified by showing that a clone selected by
antibody screening synthesized an enzyme which hydrolysed [4-
methylumbelliferoyl(p-trimethylammonium cinnamate chloride))
according to Dalrymple, B.P. et al., FEMS Microbiol. Lett.,
vol 143 (1996), 115-120.
General molecular biological techniques including DNA
isolation, restriction endonuclease digestion, ligation,
transformation as well as DNA :sequencing of the esterase gene
were performed in accordance with Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2nd. Ed. (1989), Cold Spring
Harbor, New York.
Nucleotide sequencing of the the gene encoding the enzyme
w having phenolic acid esterase activity of the present
invention was performed and the results are given in SEQ ID
N0:3. The open reading frame comprises 1608 nucleotides,
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encoding a protein of 536 amino acids with a predicted
molecular weight of 55,540 daltons.
Example 2
Measurement of pH optimum
A truncated enzyme encoded by SEQ ID NO. 1 was generated in a
PCR reaction (20 cycles of 30 seconds at 94°C, 45 seconds at
SO°C, and 1 minute at 72°C) in a buffer comprising 50 mM
Tris-
HCl, pH 9.0, 50 mM NaCl, 10 mM MgCl2, 200 ~.M dNTPs, 50
picomoles of the primers
5'-end: 5'-CGCGGATCCAACAGCGGTCCAACTGTTG-3'
3'-end: 5'-GCGAATTCTTATCTTATGGGAGAGAG-3', and
250 ng template DNA and expressed in E.coli BL21 (DE3)
(Novagen, Inc., Wisconsin, USA) using the vector pET32a
(Novagen, Inc.).
The enzyme was purified from freshly prepared cell-free
extracts by binding to Talon resin (Clontech Laboratories
Inc., California, USA) and cleaved from the metal affinity
resin using restriction grade Thrombin (Sigma) in accordance
to the guidelines provided by Novagen, Inc., USA, for use with
pET vectors. The enzyme was further purified as follows: a 1
ml MonoQ column (Pharmacia) was equilibrated with 10 mM Tris,
pH 8.0, and fresh enzyme was applied. The enzyme was eluted
at 1.0 ml/min with a sodium chloride gradient (0 to 0.5 M NaCl
in 10 mM Tris, pH 8.0). Fractions of 1.0 ml were collected.
The enzyme was assayed in McIlvaine's buffer (citric acid/
disodium hydrogen orthophosphate, see Data for Biochemical
Research, 3rd Edition, Dawson, Elliot, Elliot, Jones, Oxford
Science Publications, Oxford University Press, 1987) for pH
values ranging from 3 to 7 or a buffer comprising potassium
chloride/ boric acid for pH values ranging from 8 to 9. The
assay was carried out at 37°C with a final FAXX concentration
of 33 ACM. Ferulic acid release from FAXX was monitored
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continuously for 3 min at 335 nm according to (Faulds, C.B.
and Williamson, G., Microbiology, ~rol. 140 (1994), 779-787).
The results of the assay are give=_n in Figure 1. As can be
deduced from Figure 1, the enzyme of the invention exhibited
50o activity at about pH 5.5 and a:oout 8.5
In order to determine the temperature optimum of the enzyme
according to the invention using FAXX as a substrate, FAXX was
employed at a concentration of 33 ~.M and the assay was
performed at pH 6.0 in 100 mM MOPS buffer. The temperature of
incubation was changed from 20°C to 70°C using a
thermostatically controlled spectrophotometer. The release of
ferulic acid from FAXX was measured at 335 nm as described
above. The results are presented in Figure 2.
Kinetics
The Km, and Umax of the enzyme of. the present invention were
determined using FAXX and ArazF (0-(2-O (trans-feruloyl) -a.-
arabinofuranosyl]-(1-5)-L-arabinofuranose) as substrates. FAXX
was employed at concentrations varying from 3.72 ~,M to 49.18
~,M and Ara2F was used at concentrations ranging from 4.46 ~,M
to 122.92 ~M. The assay was performed at 37°C and pH 6.0 in
100 mM MOPS ( (3-(N-morpholino]propanesulfonic acid) ) buffer
with 90 ng enzyme. For both substrates, the release of
ferulic acid was measured at 335 nM as described above.
Based on results of the above experiments, it was determined
that the enzyme of the present invention has the following
kinetic constants:
substrate Km Umax
FAXX 3.0~0.3 ~.M 35.6~0.9 umol/min/mg
Ara2F 234~27 ~.M 19.6~1.7 ~,mol/min/mg.
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Hence, the enzyme of the present invention has a Km of about
3.0 and a Vmax of about 35 when measured under the above
conditions using FAXX as a substrate.
The specific activity of the enzyme of the present invention
was determined for methyl ferulate, methyl coumarate and
methyl p-coumarate in an assay at 37°C comprising 100 mM MOPS
buffer (with 0.020 azide), pH 6Ø, 44 ng enzyme and 1 mM of
the above substrates. After 15 minutes incubation time, the
reaction was terminated by boiling and the free acid liberated
was measured using reverse phase HPLC (Kroon, P.A. and
Williamson, G., Biotechnol. Appl. Biochem., vol. 23 (1996),
263-267). The results of the above experiment are shown
below.
In addition, the specific activity of the enzyme of the
present invention was determined for p-nitrophenyl acetate, a-
naphthyl acetate, a-naphthyl butyrate, a-naphthyl caproate cc-
naphthyl caprylate and a-naphthyl laurate according to the
methods described in Ferreira, L.M.A. et al.(Biochem. J., vol.
294 (1993), 349-355). The results of the above assay are
shown below.
substrate specific activity (U*/mg)
p-nitrophenyl acetate 204.3
a-naphthyl acetate 121
a-naphthyl butyrate 220
a-naphthyl caproate 256
a-naphthyl caprylate 54
a-naphthyl laurate 6
methyl ferulate 10.6
methyl coumarate 10.5
methyl p-coumarate 2.7
*1 U is defined as the amount of enzyme which gives 1 ~,mol/min
of ester hydrolysis.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Biotechnology and Biological Sciences Research Council
(B) STREET: Polaris House, North Star Avenue
(C) CITY: Swindon
(D) STATE:
(E) COUNTRY: United Kingdom
(F) POSTAL CODE (ZIP) : SN2 lUfi
(ii) TITLE OF INVENTION: Phenolic Acid Esterase and Us2 ThereoL
(iii) NUMBER OF SEQUENCES: 4
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/INS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID N0: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 825 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Piromyces equi
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(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..822
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
AAC AGC GGT CCA ACT GTT GAA TAC TCT ACT GAT GTT GAC TGT TCC GGT 48
Asn Ser G1y Pro Thr Val Glu Tyr Ser Thr Asp Val Asp Cys Ser Gly
1 5 10 15
A.~G ACC CTT AAG AGT AAC ACC AAC CTT AAC ATC AAT GGT CGT AAG GTT 96
Lys Thr Leu Lys Ser Asn Thr Asn Leu Asn Ile Asn G1y Arg Lys Val
20 25 30
ATT GTA AAA TTC CCA AGC GGC TTC ACT GGT GAC AAG GCT GCT CCA CTT 144
Ile Val Lys Phe Pro Ser Gly Phe Thr Gly Asp Lys Ala Ala Pro Leu
35 40 45
CTT ATT AAC TAC CAT CCA ATT ATG GGT AGT GCT TCT CAA TGG GAA AGT 192
Leu I1e Asn Tyr His Pro Ile Met Gly Ser Ala Ser Gln Trp Glu Ser
5p 55 60
GGT TCT CAA ACT GCT AAG GCT GCT TTA AAT GAT GGT GCC ATC GTT GCT 240
Gly Ser Gln Thr Ala Lys Ala Ala Leu Asn Asp Gly Ala Ile Val Ala
65 70 75 80
TTC ATG GAT GGT GCT CAA GGT CCA ATG GGA CAA GCT TGG AAC GTT GGT 288
Phe Met Asp Gly Ala Gln Gly Pro Met Gly Gln Ala Trp Asn Val Gly
85 90 95
CCA TGT TGT ACT GAT GCT GAT GAT GTT CAA TTC ACT CGT AAC TTC ATT 336
Pro Cys Cys Thr Asp Ala Asp Asp Val Gln Phe Thr Arg Asn Phe Ile
100 105 110
AAG GAA ATC ACT AGT AAG GCT TGT GTT GAT CCA AAG CGT ATC TAT GCT 384
Lys Glu Ile Thr Ser Lys Ala Cys Val Asp Pro Lys Arg Ile Tyr Ala
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IS
115 120 125
GCT GGT TTC TCT ATG GGT GGT GGT ATG TCT AAC TAT GCT GGT TGT CAA 432
Ala Gly Phe Ser Met Gly Gly Gly Met Ser Asn Tyr Ala Gly Cys G1n
130 135 140
CTT GCT GAT GTT ATT GCT GCT GCT GCT CCA TCA GCC TTT GAT CTT GCC 480
Leu Ala Asp Val Ile Ala Ala Ala Ala Pro Ser Ala Phe Asp Leu Ala
145 150 155 160
AAG GAA ATT GTT GAT GGT GGT AAA TGT AAA CCA GCT CGT CCA TTC CCA 528
Lys Glu Ile Val Asp Gly Gly Lys Cys Lys Pro Ala Arg Pro Phe Pro
165 170 175
ATC CTT AAC TTC CGT GGT ACT CAA GAT AAC GTT GTT ATG TAC AAC GGT 576
ile Leu Asn Phe Arg Gly Thr Gln Asp Asr, Val Val Met Tyr Asn Gly
180 185 190
GGT CTT TCT CAA GTT GTT CAA GGT AAG CCA ATT ACT TTC ATG GGT GCC 624
Gly Leu Ser Gln Val Val Gln Gly Lys Pro Ile Thr Phe Met Gly Ala
195 200 205
AAG AAC AAC TTC AAG GAA TGG GCT AAG ATG AAC GGA TGT ACT GGT GAA 672
Lys Asn Asn Phe Lys Glu Trp Ala Lys Met. Asn Gly Cys Thr Gly Glu
210 215 220
CCA AAA CAA AAC ACT CCA GGT AAC AAC TG'r GAA ATG TAC GAA AAC TGT 720
Pro Lys Gln Asn Thr Pro Gly Asn Asn Cya Glu Met Tyr Glu Asn Cys
225 230 235 240
AAG GGT GGT GTT AAG GTT GGT CTT TGC ACT ATC AAC GGT GGT GGT CAC 768
Lys Gly Gly Val Lys Val Gly Leu Cys Thr Ile Asn Gly Gly Gly His
245 250 255
GCT GAA GGT GAC GGT AAA ATG GGT TGG GAC TTT GTT AAA CAA TTC TCT 816
Ala Glu Gly Asp Gly Lys Met Gly Trp Asp Phe Val Lys Gln Phe Ser
260 265 270
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16
CTC CCA TAA 825
Leu Pro
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 274 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 2:
Asn Ser Gly Pro Thr Va1 Glu Tyr Ser Thr Asp Val Asp Cys Ser Gly
1 5 10 15
Lys Thr Leu Lys Ser Asn Thr Asn Leu Asn Ile Asn Gly Arg Lys Val
20 25 30
Ile Val Lys Phe Pro Ser Gly Phe Thr Gly Asp Lys Ala Ala Pro Leu
35 40 45
Leu Ile Asn Tyr His Pro Ile Met Gly Ser Ala Ser Gln Trp Glu Ser
50 55 60
Gly Ser Gln Thr Ala Lys Ala Ala Leu Asn Asp Gly Ala Ile Val Ala
65 70 75 80
Phe Met Asp Gly Ala Gln Gly Pro Met Gly Gln Ala Trp Asn Val Gly
85 90 95
Pro Cys Cys Thr Asp Ala Asp Asp Val Gln Phe Thr Arg Asn Phe Ile
100 105 110
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Lys Glu Ile Thr Ser Lys Ala Cys Val Asp Pro Lys Arg Ile Tyr Ala
115 120 125
Ala Gly Phe Ser Met Gly Gly Gly Met Ser Asn Tyr Ala Gly Cys Gln
130 135 140
Leu Ala Asp Val Ile Ala Ala Ala Ala Pro ;per Ala Phe Asp Leu Ala
145 150 155 160
Lys Glu Ile Val Asp Gly G1y Lys Cys Lys Pro Ala Arg Pro Phe Pro
165 170 175
Ile Leu Asn Phe Arg Gly Thr Gln Asp Asn Val Val Met Tyr Asn Gly
180 185 190
Gly Leu Ser Gln Val Val Gln Gly Lys Pro Ile Thr Phe Met Gly Ala
195 200 205
Lys Asn Asn Phe Lys Glu Trp Ala Lys Met Asn Gly Cys Thr Gly Glu
210 215 220
Pro Lys Gln Asn Thr Pro Gly Asn Asn Cys Glu Met Tyr Glu Asn Cys
225 230 235 240
Lys Gly Gly Val Lys Val Gly Leu Cys Thr Ile Asn Gly Gly Gly His
245 250 255
Ala Glu Gly Asp Gly Lys Met Gly Trp Asp Phe Val Lys Gln Phe Ser
260 265 270
Leu Pro
(2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1611 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Piromyces equi
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..1608
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 3:
ATG AAG ACT AGC ATT GTA TTA TCT ATC GTT GCT TTA TTT TTA ACA TCC 48
Met Lys Thr Ser Ile Val Leu Ser Ile Val Ala Leu Phe Leu Thr Ser
275 280 285 290
AAA GCT TCT GCT GAT TGT TGG TCA GAA AGA TTA GGT TGG CCA TGC TGT 96
Lys Ala Ser Ala Asp Cys Trp Ser Glu Arg Leu Gly Trp Pro Cys Cys
295 300 305
AGT GAC AGC AAT GCC GAA GTA ATC TAC GTC GAT GAC GAT GGT GAT TGG 144
Ser Asp Ser Asn Ala Glu Val Ile Tyr Val Asp Asp Asp Gly Asp Trp
310 315 320
GGT GTT GAA AAT AAT GAC TGG TGT GGT ATC CAA AAG GAA GAA GAA AAC 192
Gly Val Glu Asn Asn Asp Trp Cys Gly Ile Gln Lys Glu Glu Glu Asn
325 330 335
AAT AAC TCA TGG GAT ATG GGT GAT TGG AAC CAA GGT GGT AAC CAA GGT 240
Asn Asn Ser Trp Asp Met Gly Asp Trp Asn Gln Gly Gly Asn Gln Gly
340 345 350
GGC GGT ATG CCA TGG GGC GAC TTT GGC GGT AAC CAA GGT GGT GGT ATG 288
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Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met
355 360 365 370
CAA TGG GGT GAC TTC GGT GGT AAC CAA GGT GGC GGT ATG CCA TGG GGC 336
Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Cily Gly Met Pro Trp Gly
375 380 385
GAC TTC GGT GGT AAC CAA GGT GGC GGT ATG CCA TGG GGT GAC TTT GGC 384
Aso Phe Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly
390 395 400
GGT AAC CAA GGT GGT AAC CAA GGC GGT GGT .ATG CCA TGG GGC GAC TTT 432
Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe
405 410 415
GGA GGA AAC CAA GGA GGT AAC CAA GGT GGC GGT ATG CCA TGG GGT GAT 480
Gly Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp
420 425 430
TTC GGA GGT AAC CAA GGT GGT GGT ATG CAA TGG GGC GAC TTT GGA GGA 528
Phe Gly Gly Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly
435 440 445 450
AAC CAA GGA GGT AAC CAA GGT GGC GGT ATG CCA TGG GGT GAT TTC GGA 575
Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly
455 460 465
GGT AAC CAA GGT GGT GGT ATG CAA TGG GGC GAC TTT GGA GGA AAC CAA 624
G1y Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln
470 475 480
GGA GGT AAC CAA GGT GGC GGT ATG CCA TGG GGT GAC TTC GGA GGT AAC 672
Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn
485 490 495
CAA GGT GGT GGT ATG CAA TGG GGC GAT TTC' GGA GGT AAT CAA GGT GGT 720
Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly
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500 505 510
GGT ATG CAA TGG GGC GAC TTC GGC GGT AAC CAA GGA GGT AAC CAA GAT 768
Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Asn Gln Asp
515 520 525 530
TGG GGT AAC CAA GGT GGT AAC AGC GGT CCA ACT GTT GAA TAC TCT ACT 816
Trp Gly Asn Gln Gly Gly Asn Ser Gly Pro Thr Val Glu Tyr Ser Thr
535 540 545
GAT GTT GAC TGT TCC GGT AAG ACC CTT AAG AGT AAC ACC AAC CTT AAC 864
Asp Val Asp Cys Ser Gly Lys Thr Leu Lys Ser Asn Thr Asn Leu Asn
550 555 560
ATC AAT GGT CGT AAG GTT ATT GTA AAA TTC CCA AGC GGC TTC ACT GGT 912
Ile Asn Gly Arg Lys Val Ile Val Lys Phe Pro Ser G1y Phe Thr Gly
565 570 575
GAC AAG GCT GCT CCA CTT CTT ATT AAC TAC CAT CCA ATT ATG GGT AGT 960
Asp Lys Ala Ala Pro Leu Leu Ile Asn Tyr His Pro Ile Met Gly Ser
580 585 590
GCT TCT CAA TGG GAA AGT GGT TCT CAA ACT GCT AAG GCT GCT TTA AAT 1008
Ala Ser Gln Trp Glu 5er Gly Ser G1n Thr Ala Lys Ala Ala Leu Asn
595 600 605 610
GAT GGT GCC ATC GTT GCT TTC ATG GAT GGT GCT CAA GGT CCA ATG GGA 1056
Asp Gly Ala Ile Val Ala Phe Met Asp Gly Ala Gln Gly Pro Met Gly
615 620 625
CAA GCT TGG AAC GTT GGT CCA TGT TGT ACT GAT GCT GAT GAT GTT CAA 1104
Gln Ala Trp Asn Val Gly Pro Cys Cys Thr Asp Ala Asp Asp Val Gln
630 635 640
TTC ACT CGT AAC TTC ATT AAG GAA ATC ACT AGT AAG GCT TGT GTT GAT 1152
Phe Thr Arg Asn Phe Ile Lys Glu Ile Thr Ser Lys Ala Cys Val Asp
645 650 655
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CCA AAG CGT ATC TAT GCT GCT GGT TTC TCT ATG GGT GGT GGT ATG TCT 1200
Pro Lys Arg Ile Tyr Ala A1a Gly Phe Ser Met Gly Gly Gly Met Ser
660 665 670
AAC TAT GCT GGT TGT CAA CTT GCT GAT GTT ATT GCT GCT GCT GCT CCA 1248
Asn Tyr Ala Gly Cys Gln Leu Ala Asp Val Ile Ala Ala Ala Ala Pro
675 680 685 690
TCA GCC TTT GAT CTT GCC AAG GAA ATT GTT GAT GGT GGT AAA TGT AAA 1296
Ser Ala Phe Asp Leu Ala Lys Glu Ile Val Asp Gly Gly Lys Cys Lys
695 700 705
CCA GCT CGT CCA TTC CCA ATC CTT AAC TTC CGT GGT ACT CAA GAT AAC 1344
Pro Ala Arg Pro Phe Pro Ile Leu Asn Phe Arg Gly Thr Gln Asp Asn
710 715 720
GTT GTT ATG TAC AAC GGT GGT CTT TCT CAA GTT GTT CAA GGT AAG CCA 1392
Val Val Met Tyr Asn Gly Gly Leu Ser Gln Val Val Gln Gly Lys Pro
725 730 735
ATT ACT TTC ATG GGT GCC AAG AAC AAC TTC.' AAG GAA TGG GCT AAG ATG 1440
Ile Thr Ph2 Met Gly Ala Lys Asn Asn Phe: Lys Glu Trp Ala Lys Met
740 745 750
AAC GGA TGT ACT GGT GAA CCA AAA CAA AAC ACT CCA GGT AAC AAC TGT 1488
Asn Gly Cys Thr Gly Glu Pro Lys Gln Asn Thr Pro Gly Asn Asn Cys
755 760 765 770
GAA ATG TAC GAA AAC TGT AAG GGT GGT GT'T AAG GTT GGT CTT TGC ACT 1536
Glu Met Tyr Glu Asn Cys Lys Gly Gly Val Lys Val Gly Leu Cys Thr
775 780 785
ATC AAC GGT GGT GGT CAC GCT GAA GGT GAC GGT AAA ATG GGT TGG GAC 1584
Ile Asn Gly Gly Gly His Ala Glu Gly Asp Gly Lys Met Gly Trp Asp
790 795 800
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TTT GTT AAA CAA TTC TCT CTC CCA TAA 1611
Phe Val Lys Gln Phe Ser Leu Pro
805 810
(2) INFORMATION FOR SEQ ID N0: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 536 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Met Lys Thr Ser Ile Val Leu Ser Ile Val Ala Leu Phe Leu Thr Ser
1 5 10 15
Lvs Ala Ser Ala Asp Cys Trp Ser Glu Arg Leu Gly Trp Pro Cys Cys
20 25 30
Ser Asp Ser Asn Ala Glu Val Ile Tyr Val Asp Asp Asp Gly Asp Trp
35 40 45
Gly Val Glu Asn Asn Asp Trp Cys Gly Ile Gln Lys G1u Glu Glu Asn
50 55 60
Asn Asn Ser Trp Asp Met Gly Asp Trp Asn Gln Gly Gly Asn Gln Gly
65 70 75 80
Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met
85 90 95
G1n Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly
100 105 110
Asp Phe Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly
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115 120 125
Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe
130 135 140
Gly Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp
145 150 155 160
Phe Gly Gly Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly
165 1.70 175
Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly
180 185 190
G1y Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln
195 200 205
Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn
210 215 220
Gln Gly Gly Gly Met G1n Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly
225 230 235 240
Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Asn G1n Asp
245 250 255
Trp Gly Asn Gln Gly Gly Asn Ser Gly Pro Thr Val Glu Tyr Ser Thr
260 265 270
Asp Val Asp Cys Ser Gly Lys Thr Leu Lys Ser Asn Thr Asn Leu Asn
275 280 285
Ile Asn Gly Arg Lys Val Ile Val Lys Phe Pro Ser Gly Phe Thr G1y
290 295 300
Asp Lys Ala Ala Pro Leu Leu Ile Asn Tyr His Pro Ile Met Gly Ser
305 310 315 320
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Ala Ser Gln Trp Glu Ser Gly Ser Gln Thr Ala Lys Ala Ala Leu Asn
325 330 335
Asp Gly Ala Ile Val Ala Phe Met Asp Gly Ala Gln Gly Pro Met Gly
340 345 350
Gln Ala Trp Asn Val Gly Pro Cys Cys Thr Asp Ala Asp Asp Val Gln
355 360 365
Phe Thr Arg Asn Phe Ile Lys Glu Ile Thr Ser Lys Ala Cys Val Asp
370 375 380
Pro Lys Arg Ile Tyr Ala Ala Gly Phe Ser Met Gly Gly Gly Met Ser
385 390 395 400
Asn Tyr Ala G1y Cys Gln Leu Ala Asp Val Ile Ala Ala Ala Ala Pro
405 410 415
Ser A1a Phe Asp Leu Ala Lys Glu Ile Val Asp Gly Gly Lys Cys Lys
420 425 430
Pro Ala Arg Pro Phe Pro Ile Leu Asn Phe Arg Gly Thr Gln Asp Asn
435 440 445
Val Val Met Tyr Asn Gly Gly Leu Ser Gln Val Val Gln Gly Lys Pro
450 455 460
Ile Thr Phe Met Gly Ala Lys Asn Asn Phe Lys Glu Trp Ala Lys Met
465 470 475 480
Asn Gly Cys Thr Gly Glu Pro Lys Gln Asn Thr Pro Gly Asn Asn Cys
485 490 495
Glu Met Tyr Glu Asn Cys Lys Gly Gly Val Lys Val Gly Leu Cys Thr
500 505 510
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Ile Asn Gly Gly Gly His Ala Glu Gly Asp Gly Lys Met Gly Trp Asp
515 520 525
Phe Val Lys Gln Phe Ser Leu Pro
530 535
