U.S. patent application number 11/568848 was filed with the patent office on 2009-09-24 for novel thermostable gluconate dehydratase and use thereof.
This patent application is currently assigned to POSCO. Invention is credited to Seonghun Kim, Sun Bok Lee.
Application Number | 20090239249 11/568848 |
Document ID | / |
Family ID | 35394570 |
Filed Date | 2009-09-24 |
United States Patent
Application |
20090239249 |
Kind Code |
A1 |
Kim; Seonghun ; et
al. |
September 24, 2009 |
NOVEL THERMOSTABLE GLUCONATE DEHYDRATASE AND USE THEREOF
Abstract
The present invention relates to a novel thermostable gluconate
dehydratase from the thermoacidophilic archaeon Sulfolobus
solfataricus, a coding sequence, and an expression system. The
gluconate dehydratase has a molecular weight of about 320,000 to
380,000 daltons as the native protein, and about 40,000 to 50,000
daltons as the monomer protein, and catalyzes the dehydration
reaction of aldonic acids to 2-keto-3-deoxy derivatives at
temperatures of less than 120.degree. C. The gluconate dehydratase
can be produced from native or recombinant host cells and thereby
used in the pharmaceutical, agricultural, and other industries.
Inventors: |
Kim; Seonghun; (Ulsan,
KR) ; Lee; Sun Bok; (Pohang-city, KR) |
Correspondence
Address: |
JHK LAW
P.O. BOX 1078
LA CANADA
CA
91012-1078
US
|
Assignee: |
POSCO
Pohang-city, Kyungsangbuk-do
KR
POSTECH Foundation
Pohang-city, Kyungsangbuk-do
KR
|
Family ID: |
35394570 |
Appl. No.: |
11/568848 |
Filed: |
May 13, 2004 |
PCT Filed: |
May 13, 2004 |
PCT NO: |
PCT/KR04/01126 |
371 Date: |
November 8, 2006 |
Current U.S.
Class: |
435/29 ; 435/146;
435/232; 435/252.33; 435/320.1; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/88 20130101; C12P
7/58 20130101; C12Y 402/01039 20130101 |
Class at
Publication: |
435/29 ; 435/232;
536/23.2; 435/320.1; 435/252.33; 435/69.1; 435/146 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02; C12N 9/88 20060101 C12N009/88; C12N 15/11 20060101
C12N015/11; C12N 15/00 20060101 C12N015/00; C12N 1/21 20060101
C12N001/21; C12P 21/02 20060101 C12P021/02; C12P 7/42 20060101
C12P007/42 |
Claims
1. An isolated or purified polynucleotide encoding a gluconate
dehydratase, wherein the gluconate dehydratase comprises a
polynucleotide having at least a 50% identity to a nucleic acid
sequence encoding a polypeptide comprising amino acid sequences of
SEQ ID NO:2, or a polynucleotide complementary to the
polynucleotide having at least a 50% identity to a polynucleotide
encoding an polypeptide comprising amino acid sequences of SEQ ID
NO:2.
2. The polynucleotide according to claim 1, wherein the
polynucleotide is DNA.
3. The polynucleotide according to claim 1, wherein the
polynucleotide is RNA.
4. The polynucleotide according to claim 1, wherein the
polynucleotide comprises nucleotide sequences of SEQ ID NO:1
5. A polypeptide comprising an amino acid sequence which is at
least 50% identical to amino acid sequences of SEQ ID NO:2, wherein
the polypeptide catalyzes dehydration of aldonic acid to
2-Keto-3-deoxy aldonic acid.
6. An expression construct comprising the polynucleotide of claim
2, wherein the polynucleotide is operably linked to and under the
regulatory control of a transcription and translation regulatory
sequence.
7. An organism transformed with an expression construct according
to claim 6.
8. The organism according to claim 7, wherein the organism is
selected from the group consisting of a prokaryote, a eukaryotic
cell, and a cell derived thereof.
9. The organism according to claim 7, wherein the organism is
Escherichia coli BL21(DE3)/pGNH (KCTC10619BP).
10. A method for preparing a protein, comprising: (a) preparing a
vector comprising a polynucleotide of claim 1, operably linked to
and under the regulatory control of a transcription and translation
regulatory sequence; (b) introducing the vector into a host cell
and selecting a transformant expressing the protein; (c) culturing
the transformant under a condition which permits the protein to be
expressed; and (d) purifying the protein from the cultures, wherein
the protein catalyzes a dehydration of aldonic acid to
2-keto-3-deoxy aldonic acid.
11. A method of preparing an organism expressing a protein,
comprising: (a) preparing a vector comprising a polynucleotide of
claim 1, operably linked to and under the regulatory control of a
transcription and translation regulatory sequence; (b) introducing
the vector into a host cell; and (c) selecting a transformant
expressing the protein, wherein the protein catalyzes dehydration
of aldonic acid to 2-keto-3-deoxy aldonic acid.
12. A method of purifying a gluconate dehydratase, comprising:
conducting chromatography of a culture solution or a cell from a
gluconate dehydratase producing microorganism through a column
packed with resin attached to one or more kinds of functional
groups selected from the group consisting of carboxy,
carboxymethyl, sulpho, sulphomethyl, sulphoprophyl, aminoethyl,
diethylaminoethyl, trimethylaminomethyl, triethylaminoethyl,
dimethyl-2-hydroxyethylaminomethyl,
diethyl-2-hydroxypropylaminoethyl, phospho, alkyl and
hydroxylapatite, and wherein the matrix of the resin is selected
from the group consisting of agarose, cellulose, dextran,
polyacrylate, and polystyrene.
13. The method according to claim 12, wherein the microorganism is
thermoacidophilic archaea species.
14. The method according to claim 12, wherein the microorganism
belongs to Sulfolobus genus.
15. The method according to claim 12, wherein the microorganism is
selected from the group consisting of Sulfolobus solfataricus,
Sulfolobus acidocaldarius, Sulfolobus shibatae, Sulfolobus
tokodaii, Sulfolobus metallicus, Sulfolobus hakonensis, Sulfolobus
brierleyi, Sulfolobus islandicus, Sulfolobus tengchongensis,
Sulfolobus thuringiensis, Sulfolobus yangmingensis, Sulfolobus sp.,
Thermoplasma acidophilum, Thermoplasma volcanium, Ferroplasma
acidophilum, and Sulfolobus strains AMP12/99, CH7/99, FF5/00,
MV2/99, MVSoil3/SC2, NGB23/00, NGB6/00, NL8/00, NOB8H2, RC3,
RC6/00, or RCS1/01.
16. A method for producing a 2-keto-3-deoxy aldonic acid from
aldonic acid, comprising: contacting the gluconate dehydratase to
aldonic acid in water or an aqueous solvent at a temperature from
0.degree. C. to 120.degree. C. and a pH of 1.5 to 12, wherein the
blend ratio of a gluconate dehydratase to aldonic acid is 1 ug:
0.01 to 1 mol.
17. The method according to claim 16, wherein the gluconate
dehydratase is selected from the group consisting of an isolated
native gluconate dehydratase, a chemically synthesized gluconate
dehydratase, a recombinant gluconate dehydratase, and derivatives
thereto.
18. The method according to claim 16, wherein the aldonic acid is
selected from the group consisting of D-gluconate, D-Galactonate,
D-Galactoheptonate, D-Arabonate, D-glucuronate, L-gulonate,
D-tartarate, D-glucarate, L-isovalerate, L-threonate, D-ribonate,
L-tartarate, D-gulonate, and D-galactarate.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel nucleic acid coding
for a thermostable gluconate dehydratase from the archaeon
Sulfolobus solfataricus, a novel polypeptide coded by the nucleic
acid, and use thereof, as well as a method for preparing and
isolating the recombinant gluconate dehydratase, and catalyzing
aldonic acids to 2-keto-3-deoxy derivatives.
BACKGROUND ART
[0002] The hyperthermophilic archaea are microorganisms that grow
optimally at a temperature above 80.degree. C. Many species of
these extremely thermophilic bacteria-like organisms have been
isolated, mainly from volcanically and geothermally heated
hydrothermal environments, such as solfataric fields, hot springs,
and submarine hot vents.
[0003] The discovery of microorganisms growing optimally around
80.degree. C. is of considerable interest in both academic and
industrial communities. Both the organisms and their enzymes have
the potential to bridge the gap between biochemical catalysis and
many industrial chemical conversions. However, knowledge of the
metabolism of the hyperthermophilic microorganisms is presently
very limited.
[0004] In many hyperthermophilic archaea habited in these biotops,
the order Sulfolobales which includes the genus Sulfolobus, have a
chemolithoautotrophic metabolism which converts elemental sulfur to
hydrogen sulfide using organic compounds or hydrogen as an electron
donor. Although Sulfolobus is the sulfur-oxidizing genus, this
genus can grow chemoheterotrophically to a high cell density using
sugars. Sulfolobus solfataricus optimally grows at 80-85.degree. C.
and pH 2-4, utilizing glucose as the sole carbon and energy source
(Grogan, J. Bacteriol. 171:6710-6719, 1989)). In Sulfolobus, the
glucose metabolism pathway was first analyzed with
.sup.14C-glucose-label experiments by De Rosa et al. (Biochem. J.
224: 407-414, 1984). De Rosa's experiment shows that Sulfolobus can
convert glucose to pyruvate through a modified Entner-Doudoroff
(ED) pathway which produces non-phosphorylated intermediates such
as gluconate, 2-keto-3-deoxygluconate (KDG), and glyceraldehyde.
The first reaction of the non-phosphorylated ED pathway in S.
solfatarcus involves the NAD(P).sup.+-dependent oxidation of
glucose to gluconate, catalyzed by glucose dehydrogenase. Gluconate
is then dehydrated by gluconate dehydratase (EC 4.2.1.39) to
2-keto-3-deoxygluconate (KDG), which is cleaved to pyruvate and
glyceraldehydes, and catalyzed by KDG-alolase (EC 4.1.2.20). The
modified ED pathway involving non-phosphorylated intermediates was
also discovered in thermoacidophilic archaeon Thermoplasma
acidophilum (Budgen et al. FEBS Lett. 196:207-210, 1986). The
Thermoplasma acidophilum metabolizes glyceraldehyde formed via this
non-phosphorylated route by glyceraldehyde dehydrogenase to
glycerate, which is phosphorylated to form 2-phosphoglycerate. This
intermediate is then converted to generate one molecule of pyruvate
by enolase and pyruvate kinase. The non-phosphorylated ED pathway
is a unique glycolysis pathway discovered only in the
thermoacidophilic archaea, S. solfataricus and T. acidophilum. FIG.
1 is a non-phosphorylated ED pathway.
[0005] Another modified ED pathway involving phosphorylated
intermediates is known as a novel glycolysis route for glucose
conversion to pyruvate in some species. This metabolism was first
discovered by Szymona et al. from eubacteria Rhodobacter
sphaeroides, and was also later found from Clostridia sp. and
halobacteria (Conway, FEMS Microbiol. Rev. 103:1-28, 1992). In this
pathway, KDG produced by gluconate dehydratase is phosphorylated by
KDG kinase to 2-keto-3-deoxy-6-phosphogluconate (KDPG) and is then
cleaved by KDPG aldolase to pyruvate and
glyceraldehyde-3-phosphate. The latter intermediate is oxidized to
pyruvate, a process that involves a conventional route, via
glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase,
enolase, and pyruvate kinase.
[0006] Gluconate dehydratase has described by Kersters et al.,
Antonie van Leeuwenhoek. 37: 233-246 (1971); Kersters et al.,
Methods Enzymol. 42: 301-304 (1975); Bender et al., Eur. J.
Biochem. 40: 309-321 (1973); Bender et al., Methods Enzymol. 90:
283-287 (1982). The protein was purified and characterized only
from bacteria, Achromobacter species, and Clostridium pasteurianum,
which metabolize gluconate via a former glycolysis pathway. A
comparison of the biochemical properties of each enzymes shows that
they are very different despite in vivo the same catalytic
reaction. In thermoacidophilic archaea, S. solfataricus, and T.
acidophilum, however biochemical properties and detail mechanisms
of the gluconate dehydratases are still unknown. Despite
characterizations of two enzymes from the above-described bacteria,
no genes encoding gluconate dehydratase or partial amino acid
sequences have been reported. Hence, although recently the genomes
of S. solfataricus and T. acidophilum were completely sequenced,
putative genes encoding gluconate dehydratase could not be
annotated in the database (She et al., Proc. Natl. Acad. Sci. USA.
98: 7835-7840, 2001; Ruepp et al., Nature 407:508-513, 2000). In
addition, the known gluconate dehydratases do not maintain
thermostability at temperatures greater than about 50.degree. C.
for prolonged periods up to several hours. Thus it is necessary to
develop a novel gluconate dehydratase that can retain activity at
high temperatures for prolonged periods of time.
DISCLOSURE OF INVENTION
Technical Problem
[0007] To solve the problems of the prior art, it is an aspect of
the present invention to provide a novel thermostable gluconate
dehydratase isolated from thermoacidophilic archaea species.
[0008] It is another aspect of the present invention to provide an
amino acid sequence of protein having gluconate dehydratase
activity.
[0009] It is another aspect of the present invention to provide a
nucleic acid sequence encoding a gluconate dehydratase.
[0010] It is another aspect of the present invention to provide a
biological expression system of a gluconate dehydratase and a
transformant expressing the gluconate dehydratase.
[0011] It is another aspect of the present invention to provide an
in vitro method of conversion aldonic acid into 2-keto-3-deoxy
aldonic acid.
Technical Solution
[0012] In order to accomplish the aspects of the present invention,
the present invention provides a polynucleotide encoding a
gluconate dehydratase, wherein the gluconate dehydratase comprises
a polynucleotide having at least a 50% identity to a nucleic acid
sequence encoding an polypeptide comprising amino acid sequences of
SEQ ID NO:2 or a polynucleotide complementary to the polynucleotide
having at least a 50% identity to a polynucleotide encoding an
polypeptide comprising amino acid sequences of SEQ ID NO:2
[0013] The present invention provides a polypeptide comprising an
amino acid sequence which is at least 50% identical to an amino
acid sequence of SEQ ID NO:2, wherein the polypeptide catalyzes
dehydration of aldonic acid to 2-Keto-3-deoxy aldonic acid.
[0014] The present invention provides an expression construct
comprising a polynucleotide comprising a nucleic acid sequence
having at least a 50% identity to a nucleotide sequence encoding an
polypeptide comprising an amino acid sequence of SEQ ID NO:2 or a
polynucleotide complementary to a polynucleotide comprising a
nucleic acid sequence having at least a 50% identity to a
nucleotide sequence encoding a polypeptide comprising an amino acid
sequence of SEQ ID NO: 2, wherein the polynucleotide is operably
linked to and under the regulatory control of a transcription and
translation regulatory sequence.
[0015] The present invention provides an organism transformed with
a vector comprising a polynucleotide encoding gluconate
dehydratase, operably linked to and under the regulatory control of
a transcription and translation regulatory sequence.
[0016] The present invention provides a method for preparing a
protein, comprising:
[0017] (a) preparing a vector comprising a polynucleotide encoding
gluconate dehydratase, operably linked to and under the regulatory
control of a transcription and translation regulatory sequence;
[0018] (b) introducing the vector into a host cell and selecting a
transformant expressing the protein;
[0019] (c) culturing the transformant under a condition which
permits the protein to be expressed; and
[0020] (d) purifying the protein from intracellular material of the
transformant, and wherein the protein catalyzes a dehydration of
aldonic acid to 2-keto-3-deoxy aldonic acid.
[0021] The present invention provides a method of preparing an
organism expressing a protein, comprising:
[0022] (a) preparing a vector comprising a polynucleotide encoding
gluconate dehydratase, operably linked to and under the regulatory
control of a transcription and translation regulatory sequence;
[0023] (b) introducing the vector into a host cell; and
[0024] (c) selecting a transformant expressing the protein,
[0025] and wherein the protein catalyzes dehydration of aldonic
acid to 2-keto-3-deoxy aldonic acid.
[0026] The present invention provides a method of purifying
gluconate dehydratase, comprising:
[0027] (a) harvesting a cell from the culture solution of gluconate
dehydratase producing microorganism;
[0028] (b) obtaining a supernatant from intracellular material of
the cell;
[0029] (c) conducting chromatography of the supernatant through a
column packed with DEAE-Sepharose to collect an eluant;
[0030] (d) conducting chromatography of the eluant of step (c)
through a column packed with Q-Sepharose to collect an eluant;
[0031] (e) conducting chromatography of the eluant of step (d)
through a column packed with Phenyl-Sepharose to collect an eluant;
and
[0032] (e) conducting chromatography of the eluant of step (e)
through a Mono Q HR 5/5 column to collect a fraction.
[0033] The present invention provides a method for producing a
2-keto-3-deoxy aldonic acid from aldonic acid, comprising
contacting the gluconate dehydratase to aldonic acid in water or an
aqueous solvent at temperatures from 0.degree. C. to 120.degree. C.
and pH 1.5 to 12, wherein the blend ratio of gluconate dehydratase
to aldonic acid is 1 ug: 0.01 to 1 mol.
DESCRIPTION OF DRAWINGS
[0034] A more complete appreciation of the invention, and many of
the attendant advantages thereof, will be readily apparent as the
same becomes better understood by reference to the following
detailed description when considered in conjunction with the
accompanying drawings, wherein:
[0035] FIG. 1 is a non-phosphorylated ED pathway.
[0036] FIG. 2 is a vector map of pGNH.
[0037] FIG. 3 shows an effect of temperature on the activity of
gluconate dehydratase from S. solfataricus.
[0038] FIG. 4 shows an effect of pH on gluconate dehydratase
activity.
[0039] FIG. 5 is graph showing conversion result of 2-keo-3-deoxy
gluconate from the gluconic acid when the Ss gluconate dehydratase
was reacted to at pH 8.0 and 78.degree. C. for 6 h
MODE FOR INVENTION
[0040] In the following detailed description, only selected
embodiments of the invention have been shown and described, simply
by way of illustration of the best mode contemplated by the
inventors of carrying out the invention. As will be realized, the
invention may be modified in various respects, all without
departing from the invention. Accordingly, the drawings and
description are to be regarded as illustrative in nature, and not
restrictive.
[0041] As used herein, `purified` or isolated` refer to a nucleic
acid or polypeptide that is substantially free of cellular or viral
material with which it is naturally associated, a culture median
(when produced by recombinant DNA techniques), chemical precursors,
or other chemicals (when chemically synthesized). Moreover, an
isolated nucleic acid fragment is a nucleic acid fragment that is
not naturally occurring as a fragment and would not be found in the
natural state.
[0042] As used herein, `nucleic acid or polynucleotide` include
both RNA and DNA, including genomic DNA, cDNA, and synthetic (e.g.,
chemically synthesized) DNA. Nucleic acid can be double-stranded or
single-stranded. Where single-stranded, the nucleic acid or
polynucleotide can be a sense strand or an antisense strand. The
nucleic acid or polynucleotide can be synthesized using
oligonucleotide analogs or derivatives (e.g., inosine or
phosphorothioate nucleotides).
[0043] As used herein, `thermostable`, when referring to an enzyme,
means an enzyme which can function and is stable at high
temperatures, is heat resistant, and will not denature at high
temperatures.
[0044] A. Thermostable Gluconate Dehydratase
[0045] As used herein, the term `thermostable gluconate
dehydratase` in the context of the present invention refers to an
enzyme which:
[0046] (1) is thermostable, i.e. substantially retains enzymatic
activity upon exposure to heat at a temperature above
60-120.degree. C., preferably above 80.degree. C., and more
preferably above 90.degree. C.; and
[0047] (2) catalyzes aldonic acid to 2-keto-3-deoxy aldonic acid,
and moreover preferably reacts gluconic acid to 2-keto-3-deoxy
gluconic acid.
[0048] A gluconate dehydratase of the present invention can be
isolated or purified from the thermoacidophilic archaea species,
preferably microorganisms belong to Sulfolobus genus, and more
preferably Sulfolobus solfataricus, Sulfolobus acidocaldarius,
Sulfolobus shibatae, Sulfolobus tokodaii, Sulfolobus metallicus,
Sulfolobus hakonensis, Sulfolobus brierleyi, Sulfolobus islandicus,
Sulfolobus tengchongensis, Sulfolobus thuringiensis, Sulfolobus
yangmingensis, Sulfolobus sp., Thermoplasma acidophilum,
Thermoplasma volcanium, Ferroplasma acidophilum, or Sulfolobus
strains AMP12/99, CH7/99, FF5/00, MV2/99, MVSoil3/SC2, NGB23/00,
NGB6/00, NL8/00, NOB8H2, RC3, RC6/00, and RCS1/01.
[0049] The gluconate dehydratase of the present invention is
thermostable and maintains catalytic activity after a treatment of
about 80.degree. C. to about 90.degree. C. for 30 minutes. The
thermostable range is from 0.degree. C. to 120.degree. C.,
preferably from 20.degree. C. to 100.degree. C., and more
preferably from 30.degree. C. to 90.degree. C., and the optimum
temperature is about 85.degree. C. The gluconate dehydratase keeps
its activity in a pH range of 1.5 to 12, preferably from 1.5 to 10,
more preferably from 4.0 to 9.0, and most preferably from 6 to 8,
affording a wide range of hybridization conditions in which the
enzyme is active.
[0050] The aldonic acid as substrate for gluconate dehydratase may
include D-gluconate, D-Galactonate, D-Galactoheptonate,
D-Arabonate, D-glucuronate, L-gulonate, D-tartarate, D-glucarate,
L-isovalerate, L-threonate, D-ribonate, L-tartarate, D-gulonate,
and D-galactarate but is not limited to. The embodiment of the
present invention includes a D-gluconate as the preferred substrate
for gluconate dehydratase derived from S. solfataricus.
[0051] The gluconate dehydratase of the present invention includes
a polypeptide with biological activity that is at least about 50%,
60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical
to the amino acid sequence represented by SEQ ID NO:2. The nucleic
acid sequence of the gluconate dehydratase includes a
polynucleotide encoding polypeptide that has at least about 50%,
60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical
to the polypeptide sequence represented by SEQ ID NO:2 or its
complements. The preferable nucleic acid sequence include a
polynucleotide that is at least about 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99% identical to of SEQ ID NO:1 or its
complements. The nucleic acid sequence can further contain an
immediately contiguous sequence with both of the coding sequences
(one on the 5 end and one on the 3' end).
[0052] In one embodiment, the gluconate dehydratase from S.
solfataricus (Ss) that is designated herein as Ss gluconate
dehydratase was isolated and characterized. The Ss gluconate
dehydratase has about 320,000 to 380,000 daltons as a native form,
and has about 40,000 to 50,000 daltons as determined by SDS-PAGE
under denaturing (reducing) conditions. These results indicate that
the S. solfataricus gluconate dehydratase in its native
conformation is an octamer consisting of eight identical subunits.
The sequence of gene coding by the Ss gluconate dehydratase
includes the nucleotide sequence of SEQ ID NO:1.
[0053] B. Isolation and Purification of Thermostable Gluconate
Dehydratase
[0054] The gluconate dehydratase can be isolated and purified from
thermoacidophilic archaea species, or chemically or biochemically
synthesized by expression in a prokaryotic or eukaryotic host (for
example, by bacterial, yeast, higher plants, insects, and mammalian
cells in culture).
[0055] The purification of gluconate dehydratase can be carried out
by methods well known to those skilled in the art, i.e.,
chromatography. The chromatography can be conducted with the common
resin attached thereto, with one or more kinds of functional groups
selected from the group consisting of carboxy, carboxymethyl,
sulpho, sulphomethyl, sulphoprophyl, aminoethyl, diethylaminoethyl,
trimethyllaminomethyl, triethylaminoethyl,
dimethyl-2-hydroxyethylaminomethyl,
diethyl-2-hydroxypropylaminoethyl, phospho, alkyl (ex, hexyl-,
octyl-, phenyl-) and hyroxylapatite. The matrix of the resin can be
selected from the group consisting of agarose, cellulose, dextran,
polyacrylate, and polystyrene.
[0056] In one embodiment, the present invention provides a
purification method of gluconate dehydratase. The isolation and
purification of gluconate dehydratase is performed at below room
temperature to room temperature, preferably at about 4.degree.
C.
[0057] In the first step, the cells expressing the gluconate
dehydratase are harvested, typically by centrifugation or
filtration. In the steps, all buffers contain a stabilizing agent
or the like to increase the activity and yield of a gluconate
dehydratase preparation.
[0058] In the second step, the cells are lysed and the supernatant
is segregated and recovered from cellular debris. Lysis is
typically accomplished by mechanically applying physical stress
and/or enzymatic digestion, and segregation of the supernatant is
usually accomplished by centrifugation.
[0059] In the third step, the supernatant is further purified by
chromatography with a weak anionic exchange column. In the
embodiment, the supernatant from the second step is applied to
DEAE-Sepharose from Pharmacia (Piscataway, N.J., USA) equilibrated
with a column buffer (50 mM trihydroxymethylaminomethane (Tris), pH
7.2). The column is washed with a column buffer to remove unwanted
macromolecules, and the bound protein is then eluted off the column
with the column buffer in a linear gradient of 0-1.0 molar (M)
NaCl. In the case of Ss gluconate dehydratase, it is eluted at
about 0.5 M NaCl. The eluant fractions are collected and
centrifuged to remove any insoluble material. The collected eluant
is segregated, usually dialyzed, and then recovered to form a
fraction containing partially purified gluconate dehydratase.
[0060] In the fourth step, the fraction containing gluconate
dehydratase is further purified by chromatography with a strong
anionic exchange column. In the embodiment, the fraction is applied
to Q-Sepharose from Pharmacia (Piscataway, N.J., USA) equilibrated
with a column buffer (50 mM trihydroxymethylaminomethane (Tris), pH
7.2). The column is washed with the column buffer to remove
unwanted macromolecules, and the bound protein is then eluted off
the column with the column buffer in a linear gradient of 0-1.0
molar (M) NaCl. In the case of Ss gluconate dehydratase, it is
eluted at about 0.5 M NaCl. The eluant fractions are collected and
centrifuged to remove any insoluble material. The collected eluant
is segregated, usually dialyzed, and then recovered to form a
fraction containing partially purified Ss gluconate
dehydratase.
[0061] For increasing purity of the gluconate dehydratase, the
fraction prepared by the fourth step can be applied to a
Phenyl-Sepharose column equilibrated with 50 mM Tris-HCl, pH 7.2
containing 1.0 M NaCl. After washing with the same buffer, the
enzyme is eluted by a decreasing salt gradient of 1.0 to 0.0 M
NaCl. Active fractions, collected at a flow rate of 0.5 ml/min, are
pooled, concentrated by ultrafiltration, and loaded on a Mono Q HR
5/5 column equilibrated with 50 mM Tris-HCl, pH 7.2. The enzyme is
eluted with linear gradient of 0.0-1.0 M NaCl. Active fractions are
collected, pooled, concentrated with an ultrafiltration membrane,
and desalted with HiTrap.TM. desalting (Pharmacia, Sweden) to
eliminate remaining NaCl in enzyme fractions.
[0062] C. Identification of the Isolated and Purified Gluconate
Dehydratase and Gene Thereof
[0063] The amino acid sequence of the isolated or/and purified
gluconate dehydratase can be partially or fully determined by a
method well known in the art, such as by automated Edman
degradation, and the like. The determined amino acid sequence can
be used for screening a novel protein having homology in a database
or/and for deducing coding nucleic acids. Then, a novel gene
encoding gluconate dehydratase from various organisms can be
screened through a suitable method such as PCR, sequencing, and so
on.
[0064] The target organism may be an archaea species including
Sulfolobus solfataricus, Sulfolobus acidocaldarius, Sulfolobus
shibatae, Sulfolobus tokodaii, Sulfolobus metallicus, Sulfolobus
hakonensis, Sulfolobus brierleyi, Sulfolobus islandicus, Sulfolobus
tengchongensis, Sulfolobus thuringiensis, Sulfolobus yangmingensis,
Sulfolobus sp., Thermoplasma acidophilum, Thermoplasma volcanium,
Ferroplasma acidophilum, and Sulfolobus strains AMP12/99, CH7/99,
FF5/00, MV2/99, MVSoil3/SC2, NGB23/00, NGB6/00, NL8/00, NOB8H2,
RC3, RC6/00, and RCS1/01.
[0065] In one embodiment of the present invention, portions of the
genomic DNA encoding at least six contiguous amino acids are
synthesized and used as probes to clone full-length genes of
gluconate dehydratase. The nucleic acid encoding Ss gluconate
dehydratase and a flanked sequence thereto are identified. The open
reading frame for Ss gluconate dehydratase is shown in SEQ ID NO:1,
and the nucleic acid sequence including the 3' and 5-flanked
sequences is shown in SEQ ID NO:5.
[0066] Also, because there may not be a precisely exact match
between the nucleotide sequence in the S. solfataricus as described
herein and that in the corresponding portion of the other species
or strain, oligomers containing approximately 18 nucleotides
(encoding the six amino acid stretch) may be necessary to obtain
hybridization under conditions of sufficient stringency to
eliminate false positives.
[0067] Alternatively, polyclonal antiserum from rabbits immunized
with purified Ss gluconate dehydratase of the present invention can
be used to probe a S. solfataricus partial genomic expression
library to obtain the appropriate coding sequence.
[0068] D. Expression System of Thermostable Gluconate
Dehydratase
[0069] A gluconate dehydratase can also be produced by recombinant
DNA (rDNA) techniques. The gene encoding a thermostable gluconate
dehydratase can be operably linked to an expression system to form
an rDNA capable of expression in a compatible host. Exemplary
vectors and expression are described herein.
[0070] The gene encoding a thermostable gluconate dehydratase
includes a wild type DNA or DNA altered by modification,
substitution, deletion, or addition of nucleic acid without
substantially altering its catalytic activity or thermostability,
and such changes in sequence is acceptable and preferable where
such changes impart desirable characteristics upon the enzyme.
[0071] (1) Construction for Expression of Gluconate Dehydratase
[0072] For expression of the gluconate dehydratase, an expression
construct including a polynucleotide encoding gluconate
dehydratase, wherein the polynucleotide is operably linked to and
under the regulatory control of a transcriptional and translational
regulatory sequence, can be prepared. The transcriptional and
translational regulatory sequences are those which can function in
a specific organism (i.e., bacteria, yeast, fungi, plants, insects,
animals, and humans) cell or tissue to effect the transcriptional
and translational expression of the foreign gene with which they
are associated and can be employed according to host cell. The
examples of transcriptional and translational regulatory sequences
include a promoter, enhancer, polyadenylation signal, and
terminator, but are not limited thereto.
[0073] The promoter can be derived from a highly-expressed gene to
direct transcription of a downstream structural sequence. Such
promoters can be derived from operon encoding glycolytic enzymes
such as glyceraldehyde-3-phosphate dehydrogenase, hexokinase,
pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate
isomerase, 3-phosphoglycerate mutase, pyruvate kinase,
triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase. Other promoters that have the additional advantage of
transcription controlled by growth conditions can be employed, and
examples are alcohol dehydrogenase 2, isocytochrome C,
.alpha.-factor, acid phosphatase, heat shock proteins, degradative
enzymes associated with nitrogen metabolism, and enzymes
responsible for maltose or galactose utilization. And the promoter
may be the known promoter contained in the common vectors lacI,
lacZ, T3, T7, lamda P.sub.R, P.sub.L, trp, CMV immediate early, HSV
thymidine kinase, early and late SV40, LTRs from retroviruses, and
mouse metallothionein-I. Selection of the appropriate promoter is
well within the level of ordinary skill in the art.
[0074] The enhancer is a cis-acting elements of DNA, usually from
about 10 to 1000 bp that act on a promoter to increase its
transcription. Examples include the SV40 enhancer on the last side
of the replication origin bp 100 to 270, a cytomegalovirus early
promoter enhancer, the late side of the replication origin, and
adenovirus enhancers.
[0075] The expression construct can further include a multi-cloning
site, selectable marker, origins of replication and selectable
markers permitting transformation of the host cell, e.g., the
ampicillin resistance gene and N-terminal identification peptide
imparting desired characteristics, e.g., a sequence for stabilizing
or a simplified purification process of expressed recombinant
protein, a ribosome binding site, or/and report gene. The
expression construct may be a common vector, and examples are a
plasmid or viral vector. Large numbers of suitable vectors are
known to those of skill in the art, and are commercially available.
The following vectors are provided by way of example: pRSET,
pTrcHis, pBAD, pTOPO, pTrxFus, pThioHis (Invitrogen), pET-19, 21,
24, 32, 43 (Novagen), pQE-30, -31, -32, pQE-40, -41, -42, pQE-50,
-51, -52, pQE-16, -17, -18, pQE-60, pQE-70, pQE-9, -10, -11
(Qiagen), pBluscript II (Stratagene), pTrc99a, pKK223-3, pDR540,
pRIT2T (Amersham-Pharmacia), pXT1, pSG5 (Stratagene); pSVK3, pBPV,
pMSG, pSVLS40 (Amersham-Pharmacia), pBR322 (ATCC37017); pKK223-3
(Amersham-Pharmacia, Sweden), and pGEM1 (Promega, USA). However,
any other plasmid or vector may be used as long as they are
replicable and viable in the host.
[0076] The suitable host for producing a recombinant protein
includes a eukaryote, a prokaryote or virus. The eukaryote can be
selected from the group consisting of a yeast, insect, animal,
plant, and human, and a cell derived therefrom, and the prokaryote
can be a microorganism including E. coli, Streptomyces, Bacillus
subtilis, and fungi. Examples of the insect cell are Drosophila S2
and Spodoptera Sf9, Examples of mammalian expression systems
include the COS-7 lines of monkey kidney fibroblasts, described by
Gluzman (Cell, 23:175, 1981), and other cell lines capable of
expressing a compatible vector, for example, the C127, 3T3, CHO,
HeLa, and BHK cell lines.
[0077] (2) Establishment of Transformant
[0078] Techniques for generating transformants according to host
cell type are well known, for example calcium phosphate
transfection, DEAE-Dextran mediated transfection, electroporation
(Davis, L., Dibner, M, Battey, I., Basic Methods in Molecular
Biology, 1986), and Agrobacterium tumefaciens-mediated DNA
transfer.
[0079] In one embodiment of the present invention, pGNH vectors
harboring Ss gluconate dehydratase genes were prepared to be
introduced into Escherichia coli BL21(DE3) following select
transformants. The transformants are designed as Escherichia coli
BL21(DE3)/pGNH and been deposited pursuant to Budapest Treaty
requirements with the Korean Collection for Type Cultures (KCTC),
Taejon, Republic of Korea, in Apr. 9. 2004, and were assigned
accession number KCTC 10619BP.
[0080] The pGNH vector includes a Ss gluconate dehydratase coding
portion and control sequences at the 5 and 3' termini of the coding
portion on between BamHI and HindIII restriction sites. The
sequence of pGNH is shown in SEQ ID NO:3, and loci of each
component are represented in Table 1 and FIG. 2.
TABLE-US-00001 TABLE 1 pGNH vector Component Name loci Promoter T7
promoter 20-39 Foreign gene gluD (gluconate dehydrates 208-1396
coding gene) Selection marker Ap (ampicillin resistance 2149-2963
gene) His-tag fusion region 6xHis fusion region 100-207
[0081] (3) Production of the Recombinant Gluconate Dehydratase
[0082] Transformants are cultured in a condition for expressing the
recombinant gluconate dehydratase according to the known method.
The cultured cells employed in expression of proteins can be
disrupted by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents,
and such methods are well known to those skilled in the art. Cell
are typically harvested by centrifugation, disrupted by physical or
chemical means, and the resulting crude extract retained for
further purification.
[0083] In case of Escherichia coli BL21(DE3)/pGNH, a preferable
culture condition for expressing the recombinant Ss gluconate
dehydratase includes follows:
[0084] Medium: Luria-bertani median, M9 medium, SOB (SOC) medium,
Terrific Broth [0085] Temperature: 20-40.degree. C. [0086] Culture
time: 6-42 hrs
[0087] (4) Recover of Recombinant Protein
[0088] The recombinant gluconate dehydratase can be recovered and
purified from recombinant cell cultures by any convenient method
including ammonium sulfate precipitation, acetone precipitation,
acid extraction, anion exchange chromatography, cation exchange
chromatography, hydrophobic interaction chromatography,
phospho-cellulose chromatography, affinity chromatography,
hydroxylapatite chromatography, and lectin chromatography, and
preferably by a method of the present invention mentioned above.
Protein refolding steps can be used, as necessary, in completing
configuration of the mature protein. Finally, high performance
liquid chromatography (HPLC) can be employed for final purification
steps.
[0089] Depending upon the host employed in a recombinant production
procedure, the recombinant Ss gluconate dehydratase of the present
invention may or may not be a post-translational modification, such
as through glycosylation, phosphorylation, and acetylation. Enzymes
of the invention also may or may not include an initial methionine
amino acid residue.
[0090] In an embodiment of the present invention, recombinant Ss
gluconate dehydratase from Escherichia coli BL21(DE3)/pGNH is
purified by nickel affinity chromatography.
[0091] E. Use of Gluconate Dehydratase
[0092] The gluconate dehydratase may be employed for any purpose in
which such enzyme activity is necessary or desired. In a preferred
embodiment the enzyme is employed for catalyzing the dehydration of
aldonic acid. The dehydration of aldonic acid may be used for the
production of carbohydrate intermediates used in pharmaceutical,
agricultural, and other chemical products.
[0093] The gluconate dehydratase, their fragments, derivatives, or
analogies thereof, or recombinant gluconate dehydratase, can be
used as an immunogen to produce antibodies thereto. These
antibodies can be, for example, polyclonal or monoclonal
antibodies. The present invention also includes chimeric, single
chain, and humanized antibodies, as well as Fab fragments, and the
product of a Fab expression library. Various procedures known in
the art may be used for the production of such antibodies and
fragments.
[0094] Antibodies generated against the gluconate dehydratase can
be obtained by direct injection of the enzymes into an animal or by
administering the enzymes to an animal, preferably a nonhuman. The
antibody obtained then binds the gluconate dehydratase itself. In
this manner, even a sequence encoding only a fragment of the
gluconate dehydratase can used to generate antibodies and can then
be used to isolate the enzyme from cells expressing that gluconate
dehydratase.
[0095] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique (Kohler and
Milstein, Nature, 256:495-497, 1975), the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., Immunology Today,
4:72, 1983), and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole et al., In Monoclonal Antibodies and
Cancer Therapy, Alan R Liss, Inc., pp 77-96, 1985).
[0096] Techniques described for the production of single chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce
single chain antibodies for immunogenic enzyme products of the
present invention. Also, transgenic mice may be used to express
humanized antibodies to immunogenic enzyme products of this
invention.
[0097] Antibodies generated against the gluconate dehydratase of
the present invention may be used in screening for similar enzymes
from other organisms and samples. Antibodies may also be employed
as a probe to screen gene libraries generated from this or other
organisms to identify this or cross reactive activities.
[0098] F. Production of 2-keto-3-deoxy Aldonic Acids from Aldonic
Acids
[0099] The gluconate dehydratase dehydrates aldonic acid to
2-keto-3-deoxy aldonic acid. Thus the gluconate dehydratase of the
present invention can be used for production 2-keto-3-deoxy aldonic
acid from aldonic acid.
[0100] The present invention provides a method of producing
2-keto-3-deoxy aldonic acid from aldonic acid including contacting
the gluconate dehydratase to aldonic acid in water or an aqueous
solvent at temperatures from 0.degree. C. to 120.degree. C. and pH
1.5 to 12, wherein the blend ratio of gluconate dehydratase to
aldonic acid is 1 ug: 0.01 to 1 mol.
[0101] The gluconate dehydratase can be selected from the group
consisting of an isolated native gluconate dehydratase, a
chemically synthesized gluconate dehydratase, a recombinant
gluconate dehydratase, and derivatives thereto.
[0102] The aldonic acid prefers D-gluconate, D-Galactonate,
D-Galactoheptonate, D-Arabonate, D-glucuronate, L-gulonate,
D-tartarate, D-glucarate, L-isovalerate, L-threonate, D-ribonate,
L-tartarate, D-gulonate, and D-galactarate.
[0103] The dehydration reaction of aldonic acid is conveniently
carried out at temperatures from 0.degree. C. to 120.degree. C.,
preferably from 20.degree. C. to 100.degree. C., and most
preferably from 30.degree. C. to 90.degree. C.
[0104] The suitable pH for effecting the enzyme reaction is from
1.5 to 12, preferably from 1.5 to 10, and most preferably from 4.0
to 9.0.
[0105] The concentration of the substrate and aldonic acids in the
reaction mixture is conveniently from 1 to 700 g/L, preferably from
10 to 500 g/L, and most preferably from 50 to 200 g/L.
[0106] The optimum condition for the dehydration reaction of
aldonic acid includes the gluconate dehydratase concentration of
0.1-1 mg/mL, substrate concentration of 100-200 mM, reaction time
of less than 6 hr, temperature of 70-95.degree. C., and pH of
7.0-8.0.
[0107] The reaction is conveniently carried out in water or an
organic solvent. The organic solvent is selected from the group
consisting of alcohol, 0.01 to 100% of aqueous alcohol, and a
mixture of several alcohols, aromatic hydrocarbon, and aliphatic
hydrocarbon. The alcohol is preferably a C.sub.1-6-alkanol, such as
methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol,
or tert-butanol. The aliphatic hydrocarbon alcohol is preferably
heptane or isooctane, and the aromatic hydrocarbon alcohol is
preferably benzene or toluene. From an economic and environmental
point of view, as little organic solvent as possible is used in the
industrial process.
[0108] The dehydration reaction can be carried out in a condition
of addition of an antioxidant, such as 2-mercaptoethanol,
dithiothreitol, or cysteine, to prevent the degradation of the
produced 2-keto-3-deoxy acid analogies.
[0109] As an alternative to a gluconate dehydratase itself, the
reaction mixture may comprise an organism having gluconate
dehydratase activity.
[0110] For the reaction, any form of the gluconate dehydratase
enzyme can be used, in particular an enzyme solution, the
immobilized enzyme, intact cells of the organism having gluconate
dehydratase activity, and immobilized cells having gluconate
dehydratase activity.
[0111] The following examples are provided to further illustrate
the present invention and are not intended to limit the invention
beyond the limitations set in the appended claims.
Example 1
Cultivating sulfolobus solfataricus and Preparing Ss Cell Paste
[0112] The following describes how the hyperthermophilic archaeon
S. solfataricus is routinely grown in a 3.7 liter fermentor for the
purpose of obtaining cell mass in sufficient quantities for large
scale protein purification.
[0113] For culture maintenance, S. solfataricus P2 (DSM1617) is
routinely grown at 75-85.degree. C. as a closed shaking culture at
a volume of 100 ml. The organism was cultivated in the medium (per
liter, 3.0 g glucose, 3.0 g yeast extract, 1.3 g
(NH.sub.4).sub.2SO.sub.4, 0.28 g KH.sub.2PO.sub.4, 0.25 g
MgSO.sub.4.7H.sub.2O, 0.07 g CaCl.sub.2.H.sub.2O) containing 1 ml
trace metal solution (20 mg FeCl.sub.3.H.sub.2O, 4.5 mg
Na.sub.2B.sub.4O.sub.7.H.sub.2O, 1.8 mg MnCl.sub.2.H.sub.2O, 0.05
mg ZnSO.sub.4.H.sub.2O, 0.05 mg CuCl.sub.2.H.sub.2O, 0.04 mg
VOSO.sub.4.H.sub.2O, 0.03 mg Na.sub.2MoO.sub.4.H.sub.2O, 0.01 mg
CoSO.sub.4.H.sub.2O per liter). The final pH was adjusted to pH 3.0
with 1 M H.sub.2SO.sub.4. Cultures were grown aerobically in a
3.7-liter fermentor (KLF 2000, Bioengineering AG, Switzerland) at
78.degree. C. while being stirred at 400 rpm. Growth was monitored
spectrophotometrically at 540 nm.
Example 2
Purification of Gluconate Dehydratase from sulfolobus
solfataricus
[0114] Cells of S. solfataricus (frozen wet cell weight 35 g) were
harvested by centrifugation (5000.times.g, 30 min, 4.degree. C.)
and washed twice with 50 mM Tris-HCl (pH 7.2). Cell pellets were
re-suspended in 50 mM Tris-HCl (pH 7.2), and disrupted by
sonication for 1 h at 50% output. Crude extracts were heated at
90.degree. C. for 20 min., and heat-denatured proteins and cell
debris were removed by centrifugation (50000.times.g, 1 h,
4.degree. C.). To the supernatant solution was added solid
(NH.sub.4).sub.2SO.sub.4 up to 40% saturation to recover a fraction
containing the activity of gluconate dehydratase. After
centrifugation (50000.times.g, 1 h, 4.degree. C.), the soluble
fraction was dialyzed in 50 mM Tris-HCl (pH 7.2). The homogenate
was loaded onto a DEAE-Sepharose column (2.5.times.16 cm)
previously equilibrated with 50 mM Tris-HCl, pH 7.2, and the
elution was performed with a three bed volume of the same buffer,
followed by a linear gradient of 0.0-1.0 M NaCl. Fractions (5 ml
each) were collected at a flow rate of 1 ml/min. Those with
gluconate dehydratase activity were pooled, concentrated by
ultrafiltration on a Vivaspin.TM. concentrator membrane
(Vivascience, Lincoln, UK) and loaded on a Phenyl-Sepharose column
(1.0.times.10 cm) equilibrated with 50 mM Tris-HCl, pH 7.2,
containing 1.0 M NaCl. After washing with the same buffer, the
enzyme was eluted by a decreasing salt gradient of 1.0 to 0.0 M
NaCl. Active fractions, collected at a flow rate of 0.5 ml/min,
were pooled, concentrated by ultrafiltration, and loaded on a Mono
Q HR 5/5 column (0.5.times.5 cm) equilibrated with 50 mM Tris-HCl,
pH 7.2. The enzyme was eluted with a linear gradient of 0.0-1.0 M
NaCl. Active fractions, collected at a flow rate of 0.5 ml/min,
were pooled, concentrated with ultrafiltration membrane, and
desalted with HiTrap.TM. desalting (Pharmacia, Sweden) to eliminate
remaining NaCl in enzyme fractions.
[0115] The resulting product is referred to as Ss gluconate
dehydratase. The resultant Ss gluconate dehydratase was determined
to be 95% homogeneous by analysis of SDS-polyacrylamide gel
electrophoresis (SDS-PAGE)
Example 3
Assay of the Gluconate Dehydratase
[0116] Ss Gluconate dehydratase activity was measured by the
semicarbazide method or TBA (thiobarbituric acid) assay.
[0117] The semicarbazide method was performed as follows: an enzyme
reaction of a total volume a 400 .mu.l was incubated at 78.degree.
C. in 50 mM Tris-HCl buffer, pH 7.0, with 10 mM gluconate and an
enzyme solution. After 30 min, the enzyme reaction was stopped by
the addition of 100 .mu.l 2.0 M HCl. To this solution, 300 .mu.l of
semicarbazide solution (1.0% (w/v) semicarbazide hydrochloride and
1.5% (w/v) sodium acetate dissolved in distilled water) was added
and incubated at 30.degree. C. for 15 min. The final reaction
mixture was diluted with 500 .mu.l distilled water and then
measured at 250 nm. The absorbance coefficient of the semicarbazone
formation toward 2-keto-3-deoxy gluconate (KDG) was taken to be
0.571.times.10.sup.3 M.sup.-1 cm.sup.-1.
[0118] TBA assay was performed as follows: the reaction mixtures of
50 .mu.l were oxidized by 125 .mu.l of 25 mM periodic acid in 0.25
M H.sub.2SO.sub.4 at room temperature for 20 min. To terminate
oxidation, 250 .mu.l of 2% (w/v) sodium arsenite dissolved in 0.5 M
HCl was added to the reactants. Finally, after adding 1 ml of 0.3%
TBA to the reactants, the reaction mixtures was heated at
100.degree. C. for 10 min. Produced red chromophore was monitored
at 549 nm after adding an equal volume of DMSO. The absorbance
coefficient of thiobarbituric acid chromophore toward KDG was
estimated to be 0.347.times.10.sup.3 M.sup.-1 cm.sup.-1. One unit
of gluconate dehydratase was the amount of the enzyme producing 1
.mu.mol of 2-keto-3-deoxy gluconate per min. from gluconate under
this assay conditions. All enzyme activities were determined in
three plicate.
Example 4
Identification of the Gene Encoding Gluconate Dehydratase Through
N-Terminus Sequencing
[0119] To analyze N-terminal sequencing, purified protein was
loaded on an SDS-PAGE blotted onto a PVDF membrane, and excised.
The N-terminal sequence of gluconate dehydratase purified from S.
solfataricus was determined by Edman degradation to be MRIREIEPIV.
The deduced amino acid sequence (SEQ ID NO: 2) of gluconate
dehydratase was exactly in agreement with SSO3198, which coded for
the 45-kDa protein in the S. solfaricus P2 genome database. The
predicted protein size in the genomic database corresponded to the
single band of purified enzyme in the denaturing gel. Consequently,
this purified protein is gluconate dehydratase, and the ORF
annotated by SSO3198 is the gene, which was named gnh, encoding
gluconate de hydratase in S. solfataricus.
Example 5
Characterizing Ss Gluconate Dehydratase
[0120] 5-1. Substrate Specificities
[0121] For analysis of substrate specificities of gluconate
dehydrates, a 10 mM solution of each aldonic acid containing carbon
chains ranging from C.sub.4 to C.sub.7 were incubated together with
40 .mu.g/mL of purified protein. The amount of product formation
was measured by the semicarbazide method, which showed 100%
conversion for D-gluconate after incubation under the standard
condition. Substrate specificity of gluconate dehydratase for sugar
acids was determined by the method measuring 2-keto-3-deoxy
analogues yielded from aldonic acids. Sugar acids tested are as
follows: D-gluconate, D-galactonate, D-galactoheptonate,
D,L-arabonate, D-glucuronate, D,L-gulonate, D,L-tartarate,
D-glucarate, D,L-isovalerate, L-threonate, D-ribonate,
D-galactarate, D-xylonate, D-galacturonate, D-glucitol,
D-mannonate, and D,L-glycerate. Kinetic parameters for gluconate
dehydratase were determined using D-gluconate (0.1 to 40 mM). All
experiments were performed in three plicate.
[0122] The results of Ss gluconate dehydratase activity for the
aldonic acids are shown in Table 2. The Ss gluconate dehydratase
showed higher selectivity to D-gluconate than any other adonic
acids. D-Galactonate and D-galactoheptonate could be used as
substrates for the enzyme. Negligible but detectable activities
(less than 1% of activity toward D-gluconate) were observed for the
following substrates: D-glucuronate, L-gulonate, D-tartarate,
D-glucarate, Et L-isovalerate, L-threonate, D-ribonate,
L-tartarate, D-gulonate, and D-galactarate. It therefore appears
that the enzyme has a preference to D-gluconate.
TABLE-US-00002 TABLE 2 Relative Probable structure of Substrates
activity (%) dehydration products D-Gluconate 100.0
2-keto-3-deoxy-D-gluconate D-Galactonate 2.8
2-keto-3-deoxy-D-galactonate D-Galactoheptonate 1.6
2-keto-3-deoxy-D-galactoheptonate Substrates Relative Probable
structure of dehydration activity (%) products D-Arabonate 0.7
2-keto-4,5-dihydroxy-D-valeric acid Less than 1% activity on the
following substrates; D-glucuronate (0.65), L-gulonate (0.41),
D-tartarate (0.41), D-glucarate (0.32) D,L-isovalerate (0.25),
L-threonate (0.16), D-ribonate (0.16), L-tartarate (0.16),
D-gulonate (0.16), and D-galactarate (0.10). No reaction on the
following substrates: L-arabonate, D-xylonate, D-galacturonate,
D-glucitol, D-mannonate, and D,L-glycerate. The relative enzyme
activity was assayed by measuring the 2-keto-3-deoxy analogues
produced from 10 mM each of aldonic acid containing 1 mM CoCl2 in
50 mM Tri-HCl buffer (pH 7.0) for 30 min at 78?C. using the
semicarbazide method.
[0123] Biochemical and kinetic parameters for the enzyme were
determined using the assay method described above under standard
conditions.
[0124] 5-2. Kinetic Parameters
[0125] Values for V.sub.max and K.sub.m were determined from
Lineweaver bulk plots. The rate dependence on substrate
concentration followed Michaelis-Meten kinetics. From
Lineweaver-Burk plots, K.sub.m and V.sub.m values of 16.7 mM and
34.5 units/mg were determined with D-gluconate as the substrate.
The turnover number (k.sub.cat) was cat calculated as 333 s.sup.-1
for gluconate dehydratase, and the value of k.sub.cat/K.sub.m was
19.9.
[0126] 5-3. Optimum Temperature
[0127] The temperature profile for enzyme activity was determined
between 40 and 100.degree. C. FIG. 3 shows an effect of temperature
on the activity of gluconate dehydratase from S. solfataricus. The
purified gluconate dehydratase displayed optimal activity between
80 and 90.degree. C. Enzyme activity was not detectable below
60.degree. C.
[0128] 5-4. Thermostability
[0129] Enzyme thermostability was determined at 80, 90, and
100.degree. C. by incubating enzyme solution (50 .mu.g/ml) in 50 mM
Tris-HCl (pH 7.2). At an appropriate time, samples were taken and
completely cooled on ice and then measured for residual activities
under standard conditions. The thermostability of purified
gluconate dehydrates was measured at 80, 90, and 100.degree. C. At
80.degree. C., the optimal temperature for growth of S.
solfataricus P2, the gluconate dehydratase was very stable over 2
hours. At 90.degree. C., enzyme activity decreased below 50% after
a 2 hour incubation. At 100.degree. C., however, the enzyme had a
half-life of less than 40 min.
[0130] 5-5. Optimum pH
[0131] The effect of pH on gluconate dehydratase activity was
determined at 78.degree. C. in a citric acid-NaOH buffer (pH
2.7-5.0), 50 mM Tris-HCl buffer (pH 5.8-8.0), and 50 mM
glycine-NaOH buffer (pH 8.5-10.5).
[0132] FIG. 4 shows an effect of pH on gluconate dehydratase
activity; 50 mM citric acid-NaOH (.box-solid.), 50 mM Tris-HCl ( ),
and 50 mM glycine-NaOH buffer (.largecircle.). In FIG. 4, within
the pH range from pH 2.7 to pH 10.5, the activity of purified
enzyme displayed an optimum between pH 7.0 to 8.0.
Example 6
Mass Determination of Native Gluconate Dehydratase
[0133] Pure Ss gluconate dehydratase (100 .mu.g) of EXAMPLE 2 was
chromatogramed through a Sephacryl S-200 column (1.0.times.89 cm)
using the gel filtration calibration kit (Pharmacia Biotech,
Sweden). The equilibrium and elution buffer used was 50 mM
Tris-HCl, pH 7.2, containing 150 mM NaCl, and the flow rate was 0.5
ml/min. The molecular weight markers used were thyroglobulin (669
kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa),
BSA (67 kDa), ovalbumin (43 kDa), chymotrypsin (25 kDa), and
ribonuclease A (13.7 kDa). Proteins were detected at 280 nm, and
gluconate dehydratase activity was measured by the standard method.
The gluconate dehydratase molecular weight was calculated by
interpolation on a plot of log molecular mass against the K.sub.av
values following the recommended procedure.
[0134] The native molecular weight of purified enzyme was 357.+-.42
kDa, as measured on a calibrated Sephacryl S-200 column with
standard molecular weight markers. The molecular mass of
denaturated gluconate dehydratase determined from SDS-PAGE was
approximately 44 kDa. These results indicate that the S.
solfataricus gluconate dehydratase in its native conformation is an
octamer consisting of eight identical subunits.
Example 7
Cloning of Gene Coding Gluconate Dehydratase from Sulfolobus
solfataricus
[0135] 7-1. Cloning
[0136] The gene coding thermostable gluconate dehydratase was
cloned from the hyperthermophilic archaeon Sulfolobus solfataricus
(Ss).
[0137] Amino terminal protein microsequencing was performed by the
Korea Basic Science Institute (KBSI) (Daejeon, Korea) on 100
picomoles (pmol) of homogeneous native Ss gluconate dehydratase
prepared as described in Example 2. The sequence of the 10
N-terminal amino acid residues thereby obtained was later shown to
correspond exactly with deduced residues shown in SEQ ID NO 4 from
residue 1 to residue 10.
[0138] DNA encoding the Ss gluconate dehydratase of the present
invention, SEQ ID NO 1, was initially amplified from Sulfolobus
solfataricus genomic DNA by the PCR technique using the primer set
of SEQ ID NO:5 and 6, including the BamHI restriction site and
HindIII restriction site. The amplified fragments were inserted
into the BamHI and HindIII sites of pGEM-T easy (Promega, USA) and
the resulting vector was digested by each BamHI and HindIII
restriction enzyme. The 1,188 bp fragments were ligated into the
BamHI and HindIII sites of pRSET vector (Invitrogen, USA) including
antibiotic resistance (Amp.sup.r), a bacterial origin of
replication (ori), and IPTG-regulatable promoter operator (P/O), a
ribosome binding site (RBS), a 6-His tag, and restriction enzyme
sites, and the resulting vector was designated as pGNH. The pGNH
contains the complete 3,993 bp fragment encoding Ss gluconate
dehydratase flanked at the fragment's termini by BamHI and
HindIII.
[0139] The pGNH was then used to transform the E. coli strain
BL21(DE3) which is a protease-deficient mutant to protect
heterologously expressed proteins against protease. Transformants
were selected by growing in LB medium supplemented with ampicillin,
and were harvested to confirmed whether the gnh gene was placed
therein by restriction analysis.
[0140] 7-2. Expression
[0141] Transformants were grown overnight in a liquid culture in LB
media supplemented with Amp (100 .mu.g/ml). The overnight culture
was used to inoculate a large culture at a ratio of 1:100 to 1:250.
The cells were grown to an optical density (OD.sub.600) of between
0.4 and 0.6. Isopropyl-.beta.-D-thiogalactopyranoside (IPTG) was
then added to a final concentration of 1 mM IPTG induces by
inactivating the lac repressor, clearing the P/O leading to
increased gene expression. Cells were grown an extra 4 to 6 hours,
and were then harvested by centrifugation.
Example 8
Purification of Recombinant Ss Gluconate Dehydratase
[0142] Recombinant Ss gluconate dehydrates was purified from E.
coli containing the plasmid pGNH described in Example 7.
[0143] Cultures of Escherichia coli BL21(DE3)/pGNH were prepared as
before, and 30 grams of cultured cells were isolated, admixed in
120 ml lysis buffer (prepared as in Example 2), and sonicated 10
times for 6 minutes each at full power. The resulting lysate was
centrifuged for 30 minutes at 7,000 rpm. The supernatant from
centrifugation was isolated and then placed for 20 minutes in a
90.degree. C. water bath. The heat-denaturated solution was then
centrifuged as above and the resultant was isolated and then loaded
on an IMAC.sup.? column equilibrated in 50 mM Tris-HCl, pH 7.2 as
described in Example 2. The column was washed with 3 column volumes
of the same buffer, and then eluted with a gradient of 0-0.2 M
imidazole in the same buffer, thereby collecting gradient elution
fractions. The gluconate dehydratase activity assay was performed
on each fraction, and peak activity fractions were pooled and
dialyzed in 50 mM Tris-HCl (pH 7.2).
[0144] Following dialysis, the dialysate was loaded on a
Q-Sepharose column equilibrated with 50 mM Tris-HCl, pH 7.2, as
described in Example 2. The column was washed with 3 column volumes
and eluted with a 0-1.0 M NaCl gradient in 50 mM Tris-HCl (pH 7.2).
Peak activity fractions were pooled and assayed, and active
fractions were pooled and concentrated 10-20 fold in a Vivaspin.TM.
concentrator (Vivascience, Lincoln, UK). The concentrated pool was
then dialyzed against a final dialysis buffer to form purified
recombinant Ss gluconate dehydratase.
[0145] The activity of the recombinant Ss gluconate dehydratase was
determined by the method described in Example 3.
Example 9
Dehydration of Gluconic Acid to 2-Keto-3-deoxygluconate by
Recombinant Gluconate Dehydratase
[0146] The recombinant gluconate dehydratase from S. solfaricus was
used for the dehydration of gluconic acid to 2-keto-3-deoxy
gluconate.
[0147] The reaction mixture consisted of 1, 5, 10, 50, and 100 mM
gluconic acid sodium salt (Sigma Chemical Co., St. Louis, Mo.,
USA), and the Ss gluconate dehydratase in 50 mM Tris-HCl buffer (pH
8.0). The gluconate dehydratase was added at a concentration of 3.5
mg/ml, and the reaction was carried out at 78.degree. C. for 6
hours. 2-Keto-3-deoxy gluconate was assayed by the standard
procedure described in Example 3. 2-Keto-3-deoxy gluconate was
produced by the Ss gluconate dehydratase as shown in FIG. 5.
[0148] The optimum conditions of recombinant Ss gluconate
dehydratase for dehydrating aldonic acid to 2-keto-3-deoxy aldonic
acid follow:
[0149] Enzyme concentration: 0.1-1 mg/mL
[0150] Substrate concentration: 100-200 mM
[0151] Reaction time: within 6 hours
[0152] Temperature: about 80.degree. C.
[0153] pH: 7.5-8.0
Sequence List Text
[0154] SEQ ID NO:1--Open Reading Frame encoding gluconate
dehydratase from Sulfolobus solfataricus.
[0155] SEQ ID NO:2--amino acid sequence of gluconate dehydratase
from Sulfolobus solfataricus.
[0156] SEQ ID NO:3--nucleic acid sequence of pGNH vector.
[0157] SEQ ID NO:4--N-terminal amino acid sequence from the
gluconate dehydratase purified from Sulfolobus solfataricus.
[0158] SEQ ID NO:5--nucleic acid sequence of sense primer with
BamHI restriction site.
[0159] SEQ ID NO:6--nucleic acid sequence of antisense primer with
HindIII restriction site.
Sequence CWU 1
1
611189DNASulfolobus solfataricusCDS(1)..(1185)Open Reading Frame
encoding gluconate dehydratase 1atg aga atc aga gaa ata gaa cca ata
gta ctc acc tcg aaa gag aaa 48Met Arg Ile Arg Glu Ile Glu Pro Ile
Val Leu Thr Ser Lys Glu Lys1 5 10 15gga agt gca act tgg gca tct ata
atg att gtc aca agg gtc att acg 96Gly Ser Ala Thr Trp Ala Ser Ile
Met Ile Val Thr Arg Val Ile Thr 20 25 30gaa aat ggg gaa gta ggc tat
ggt gag gca gta ccc aca cta aga gtt 144Glu Asn Gly Glu Val Gly Tyr
Gly Glu Ala Val Pro Thr Leu Arg Val 35 40 45ata tct gta tat aac gca
att aaa caa gtt agt aag gct tat ata ggg 192Ile Ser Val Tyr Asn Ala
Ile Lys Gln Val Ser Lys Ala Tyr Ile Gly 50 55 60aaa gag gta gag gaa
gtt gag aag aac tat cat gaa tgg tat aaa caa 240Lys Glu Val Glu Glu
Val Glu Lys Asn Tyr His Glu Trp Tyr Lys Gln65 70 75 80gat ttc tat
tta gct agg tct ttt gaa tca gca act gca gta agt gca 288Asp Phe Tyr
Leu Ala Arg Ser Phe Glu Ser Ala Thr Ala Val Ser Ala 85 90 95atc gat
ata gcc tca tgg gat ata ata ggg aaa gag ctt gga gca cca 336Ile Asp
Ile Ala Ser Trp Asp Ile Ile Gly Lys Glu Leu Gly Ala Pro 100 105
110att cat aaa tta tta gga gga aaa acc agg gat agg gta cca gtc tac
384Ile His Lys Leu Leu Gly Gly Lys Thr Arg Asp Arg Val Pro Val Tyr
115 120 125gca aac gga tgg tat cag gac tgc gta act cca gag gaa ttt
gcg gaa 432Ala Asn Gly Trp Tyr Gln Asp Cys Val Thr Pro Glu Glu Phe
Ala Glu 130 135 140aag gca aaa gac gtt gta aag atg gga tat aag gct
tta aaa ttt gat 480Lys Ala Lys Asp Val Val Lys Met Gly Tyr Lys Ala
Leu Lys Phe Asp145 150 155 160ccg ttt ggt cca tat tac gat tgg ata
gat gag aga ggt cta aga gaa 528Pro Phe Gly Pro Tyr Tyr Asp Trp Ile
Asp Glu Arg Gly Leu Arg Glu 165 170 175gct gag gag aga gta aag gct
gtt aga gag gca gtt gga gac aac gtg 576Ala Glu Glu Arg Val Lys Ala
Val Arg Glu Ala Val Gly Asp Asn Val 180 185 190gat att tta ata gag
cat cac ggt agg ttt aat gcg aat tcg gct att 624Asp Ile Leu Ile Glu
His His Gly Arg Phe Asn Ala Asn Ser Ala Ile 195 200 205atg ata gcg
aaa aga ttg gaa aaa tac aat ccg gga ttt atg gag gaa 672Met Ile Ala
Lys Arg Leu Glu Lys Tyr Asn Pro Gly Phe Met Glu Glu 210 215 220ccg
gta cat cat gag gac gta att ggt tta aga aag tat aaa gcc agt 720Pro
Val His His Glu Asp Val Ile Gly Leu Arg Lys Tyr Lys Ala Ser225 230
235 240act cat tta agg gtt gca ttg gga gaa aga ctg ata agt gaa aag
gaa 768Thr His Leu Arg Val Ala Leu Gly Glu Arg Leu Ile Ser Glu Lys
Glu 245 250 255act gcg ttt tac gtt gag gaa ggt ctt gta aac ata ttg
caa cca gat 816Thr Ala Phe Tyr Val Glu Glu Gly Leu Val Asn Ile Leu
Gln Pro Asp 260 265 270tta act aat ata ggt ggt gta aca gta ggt agg
agt gtt ata aaa ata 864Leu Thr Asn Ile Gly Gly Val Thr Val Gly Arg
Ser Val Ile Lys Ile 275 280 285gct gaa gct aat gat gta gag gtg gct
ttt cac aac gcc ttt ggt tca 912Ala Glu Ala Asn Asp Val Glu Val Ala
Phe His Asn Ala Phe Gly Ser 290 295 300ata cag aat gca gtt gaa ata
caa cta agt gca gtt aca cag aat ttg 960Ile Gln Asn Ala Val Glu Ile
Gln Leu Ser Ala Val Thr Gln Asn Leu305 310 315 320tat tta ctt gag
aac ttc tat gat tgg ttc cct cag tgg aaa agg gat 1008Tyr Leu Leu Glu
Asn Phe Tyr Asp Trp Phe Pro Gln Trp Lys Arg Asp 325 330 335tta gta
tat aat gaa acg cca gtt gaa gga ggt cac gtt aag gtt cca 1056Leu Val
Tyr Asn Glu Thr Pro Val Glu Gly Gly His Val Lys Val Pro 340 345
350tac aag cct gga cta ggt gtt tca att aat gaa aaa ata ata gaa cag
1104Tyr Lys Pro Gly Leu Gly Val Ser Ile Asn Glu Lys Ile Ile Glu Gln
355 360 365cta aga gct gaa cca ata cca tta gat gta att gaa gaa ccg
gtt tgg 1152Leu Arg Ala Glu Pro Ile Pro Leu Asp Val Ile Glu Glu Pro
Val Trp 370 375 380gtc gtc aag gga acc tgg aag aat tat ggt gtt tgaa
1189Val Val Lys Gly Thr Trp Lys Asn Tyr Gly Val385 390
3952395PRTSulfolobus solfataricus 2Met Arg Ile Arg Glu Ile Glu Pro
Ile Val Leu Thr Ser Lys Glu Lys1 5 10 15Gly Ser Ala Thr Trp Ala Ser
Ile Met Ile Val Thr Arg Val Ile Thr 20 25 30Glu Asn Gly Glu Val Gly
Tyr Gly Glu Ala Val Pro Thr Leu Arg Val 35 40 45Ile Ser Val Tyr Asn
Ala Ile Lys Gln Val Ser Lys Ala Tyr Ile Gly 50 55 60Lys Glu Val Glu
Glu Val Glu Lys Asn Tyr His Glu Trp Tyr Lys Gln65 70 75 80Asp Phe
Tyr Leu Ala Arg Ser Phe Glu Ser Ala Thr Ala Val Ser Ala 85 90 95Ile
Asp Ile Ala Ser Trp Asp Ile Ile Gly Lys Glu Leu Gly Ala Pro 100 105
110Ile His Lys Leu Leu Gly Gly Lys Thr Arg Asp Arg Val Pro Val Tyr
115 120 125Ala Asn Gly Trp Tyr Gln Asp Cys Val Thr Pro Glu Glu Phe
Ala Glu 130 135 140Lys Ala Lys Asp Val Val Lys Met Gly Tyr Lys Ala
Leu Lys Phe Asp145 150 155 160Pro Phe Gly Pro Tyr Tyr Asp Trp Ile
Asp Glu Arg Gly Leu Arg Glu 165 170 175Ala Glu Glu Arg Val Lys Ala
Val Arg Glu Ala Val Gly Asp Asn Val 180 185 190Asp Ile Leu Ile Glu
His His Gly Arg Phe Asn Ala Asn Ser Ala Ile 195 200 205Met Ile Ala
Lys Arg Leu Glu Lys Tyr Asn Pro Gly Phe Met Glu Glu 210 215 220Pro
Val His His Glu Asp Val Ile Gly Leu Arg Lys Tyr Lys Ala Ser225 230
235 240Thr His Leu Arg Val Ala Leu Gly Glu Arg Leu Ile Ser Glu Lys
Glu 245 250 255Thr Ala Phe Tyr Val Glu Glu Gly Leu Val Asn Ile Leu
Gln Pro Asp 260 265 270Leu Thr Asn Ile Gly Gly Val Thr Val Gly Arg
Ser Val Ile Lys Ile 275 280 285Ala Glu Ala Asn Asp Val Glu Val Ala
Phe His Asn Ala Phe Gly Ser 290 295 300Ile Gln Asn Ala Val Glu Ile
Gln Leu Ser Ala Val Thr Gln Asn Leu305 310 315 320Tyr Leu Leu Glu
Asn Phe Tyr Asp Trp Phe Pro Gln Trp Lys Arg Asp 325 330 335Leu Val
Tyr Asn Glu Thr Pro Val Glu Gly Gly His Val Lys Val Pro 340 345
350Tyr Lys Pro Gly Leu Gly Val Ser Ile Asn Glu Lys Ile Ile Glu Gln
355 360 365Leu Arg Ala Glu Pro Ile Pro Leu Asp Val Ile Glu Glu Pro
Val Trp 370 375 380Val Val Lys Gly Thr Trp Lys Asn Tyr Gly Val385
390 39534050DNASulfolobus solfataricusgene(208)..(1396)Open Reading
Frame encoding gluconate dehydratase from Sulfolobus solfataricus
3gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct
60agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat
120catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga
tctgtacgac 180gatgacgata aggatcgatg gggatccatg agaatcagag
aaatagaacc aatagtactc 240acctcgaaag agaaaggaag tgcaacttgg
gcatctataa tgattgtcac aagggtcatt 300acggaaaatg gggaagtagg
ctatggtgag gcagtaccca cactaagagt tatatctgta 360tataacgcaa
ttaaacaagt tagtaaggct tatataggga aagaggtaga ggaagttgag
420aagaactatc atgaatggta taaacaagat ttctatttag ctaggtcttt
tgaatcagca 480actgcagtaa gtgcaatcga tatagcctca tgggatataa
tagggaaaga gcttggagca 540ccaattcata aattattagg aggaaaaacc
agggataggg taccagtcta cgcaaacgga 600tggtatcagg actgcgtaac
tccagaggaa tttgcggaaa aggcaaaaga cgttgtaaag 660atgggatata
aggctttaaa atttgatccg tttggtccat attacgattg gatagatgag
720agaggtctaa gagaagctga ggagagagta aaggctgtta gagaggcagt
tggagacaac 780gtggatattt taatagagca tcacggtagg tttaatgcga
attcggctat tatgatagcg 840aaaagattgg aaaaatacaa tccgggattt
atggaggaac cggtacatca tgaggacgta 900attggtttaa gaaagtataa
agccagtact catttaaggg ttgcattggg agaaagactg 960ataagtgaaa
aggaaactgc gttttacgtt gaggaaggtc ttgtaaacat attgcaacca
1020gatttaacta atataggtgg tgtaacagta ggtaggagtg ttataaaaat
agctgaagct 1080aatgatgtag aggtggcttt tcacaacgcc tttggttcaa
tacagaatgc agttgaaata 1140caactaagtg cagttacaca gaatttgtat
ttacttgaga acttctatga ttggttccct 1200cagtggaaaa gggatttagt
atataatgaa acgccagttg aaggaggtca cgttaaggtt 1260ccatacaagc
ctggactagg tgtttcaatt aatgaaaaaa taatagaaca gctaagagct
1320gaaccaatac cattagatgt aattgaagaa ccggtttggg tcgtcaaggg
aacctggaag 1380aattatggtg tttgaaagct tgatccggct gctaacaaag
cccgaaagga agctgagttg 1440gctgctgcca ccgctgagca ataactagca
taaccccttg gggcctctaa acgggtcttg 1500aggggttttt tgctgaaagg
aggaactata tccggatctg gcgtaatagc gaagaggccc 1560gcaccgatcg
cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta
1620gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
acacttgcca 1680gcgccctagc gcccgctcct ttcgctttct tcccttcctt
tctcgccacg ttcgccggct 1740ttccccgtca agctctaaat cgggggctcc
ctttagggtt ccgatttagt gctttacggc 1800acctcgaccc caaaaaactt
gattagggtg atggttcacg tagtgggcca tcgccctgat 1860agacggtttt
tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
1920aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
gggattttgc 1980cgatttcggc ctattggtta aaaaatgagc tgatttaaca
aaaatttaac gcgaatttta 2040acaaaatatt aacgcttaca atttaggtgg
cacttttcgg ggaaatgtgc gcggaacccc 2100tatttgttta tttttctaaa
tacattcaaa tatgtatccg ctcatgagac aataaccctg 2160ataaatgctt
caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
2220ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
aaacgctggt 2280gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
ggttacatcg aactggatct 2340caacagcggt aagatccttg agagttttcg
ccccgaagaa cgttttccaa tgatgagcac 2400ttttaaagtt ctgctatgtg
gcgcggtatt atcccgtatt gacgccgggc aagagcaact 2460cggtcgccgc
atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
2520gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
ccatgagtga 2580taacactgcg gccaacttac ttctgacaac gatcggagga
ccgaaggagc taaccgcttt 2640tttgcacaac atgggggatc atgtaactcg
ccttgatcgt tgggaaccgg agctgaatga 2700agccatacca aacgacgagc
gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 2760caaactatta
actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
2820ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
gctggtttat 2880tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
atcattgcag cactggggcc 2940agatggtaag ccctcccgta tcgtagttat
ctacacgacg gggagtcagg caactatgga 3000tgaacgaaat agacagatcg
ctgagatagg tgcctcactg attaagcatt ggtaactgtc 3060agaccaagtt
tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
3120gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
gtgagttttc 3180gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
tcttcttgag atcctttttt 3240tctgcgcgta atctgctgct tgcaaacaaa
aaaaccaccg ctaccagcgg tggtttgttt 3300gccggatcaa gagctaccaa
ctctttttcc gaaggtaact ggcttcagca gagcgcagat 3360accaaatact
gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
3420accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
gtggcgataa 3480gtcgtgtctt accgggttgg actcaagacg atagttaccg
gataaggcgc agcggtcggg 3540ctgaacgggg ggttcgtgca cacagcccag
cttggagcga acgacctaca ccgaactgag 3600atacctacag cgtgagctat
gagaaagcgc cacgcttccc gaagggagaa aggcggacag 3660gtatccggta
agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
3720cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
gtcgattttt 3780gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
agcaacgcgg cctttttacg 3840gttcctggcc ttttgctggc cttttgctca
catgttcttt cctgcgttat cccctgattc 3900tgtggataac cgtattaccg
cctttgagtg agctgatacc gctcgccgca gccgaacgac 3960cgagcgcagc
gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
4020ccccgcgcgt tggccgattc attaatgcag 4050410PRTSulfolobus
solfataricusPEPTIDE(1)..(10)N-terminal amino acid sequence from the
gluconate dehydratase purified from Sulfolobus solfataricus 4Met
Arg Ile Arg Glu Ile Glu Pro Ile Val1 5 10530DNAArtificial
Sequencesense primer with BamHI restriction site 5cgggatccat
gagaatcaga gaaatagaac 30628DNAArtificial Sequenceantisense primer
with HindIII restriction site 6cccaagcttt caaacaccat aattacag
28
* * * * *