U.S. patent application number 11/184834 was filed with the patent office on 2006-01-26 for glucose dehydrogenase.
Invention is credited to Koji Sode.
Application Number | 20060019328 11/184834 |
Document ID | / |
Family ID | 26460990 |
Filed Date | 2006-01-26 |
United States Patent
Application |
20060019328 |
Kind Code |
A1 |
Sode; Koji |
January 26, 2006 |
Glucose dehydrogenase
Abstract
Modified water-soluble glucose dehydrogenases having
pyrrolo-quinoline quinone as a coenzyme are provided wherein at
least one amino acid residue is replaced by another amino acid
residue in a specific region. Modified water-soluble PQQGDHs of the
present invention have improved affinity for glucose.
Inventors: |
Sode; Koji; (Tokyo,
JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
26460990 |
Appl. No.: |
11/184834 |
Filed: |
July 20, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09959549 |
Oct 30, 2001 |
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PCT/JP00/02872 |
May 1, 2000 |
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11184834 |
Jul 20, 2005 |
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Current U.S.
Class: |
435/26 ; 435/189;
435/252.3; 435/471; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/0006
20130101 |
Class at
Publication: |
435/026 ;
435/069.1; 435/189; 435/252.3; 435/471; 536/023.2 |
International
Class: |
C12Q 1/32 20060101
C12Q001/32; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C12N 9/02 20060101 C12N009/02; C12N 15/74 20060101
C12N015/74; C12N 1/21 20060101 C12N001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 30, 1999 |
JP |
124285/1999 |
Jan 18, 2000 |
JP |
9137/2000 |
Claims
1. An isolated modified water-soluble glucose dehydrogenase having
pyrroloquinoline quinone as a coenzyme characterized in that at
least one amino acid residue in a natural water-soluble glucose
dehydrogenase derived from Acinetobacter is replaced by another
amino acid residue and it has improved affinity for glucose as
compared with the natural water-soluble glucose dehydrogenase.
2. The isolated modified glucose dehydrogenase of claim 1 having
high selectivity for glucose as compared with the wild-type PQQGDH
of SEQ ID NO: 1.
3. An isolated modified glucose dehydrogenase having
pyrrolo-quinoline quinone as a coenzyme wherein asparagine 462
(asparagine 438 of SEQ ID NO: 1) in the water-soluble PQQGDH
derived from Acinetobacter calcoaceticus or an amino acid residue
corresponding to said residue is replaced by another amino acid
residue.
4. An isolated modified glucose dehydrogenase having
pyrrolo-quinoline quinone as a coenzyme wherein asparagine 452
(asparagine 428 of SEQ ID NO: 1) in the water-soluble PQQGDH
derived from Acinetobacter calcoaceticus or an amino acid residue
corresponding to said residue is replaced by another amino acid
residue.
5. An isolated modified glucose dehydrogenase having
pyrrolo-quinoline quinone as a coenzyme wherein lysine 455 (lysine
431 of SEQ ID NO: 1) in the water-soluble PQQGDH derived from
Acinetobacter calcoaceticus or an amino acid residue corresponding
to said residue is replaced by another amino acid residue.
6. An isolated modified glucose dehydrogenase having
pyrrolo-quinoline quinone as a coenzyme wherein aspartate 456
(aspartate 432 of SEQ ID NO: 1) in the water-soluble PQQGDH derived
from Acinetobacter calcoaceticus or an amino acid residue
corresponding to said residue is replaced by another amino acid
residue.
7. An isolated modified glucose dehydrogenase having
pyrrolo-quinoline quinone as a coenzyme wherein aspartate 457
(aspartate 433 of SEQ ID NO: 1) in the water-soluble PQQGDH derived
from Acinetobacter calcoaceticus or an amino acid residue
corresponding to said residue is replaced by another amino acid
residue.
8. An isolated modified glucose dehydrogenase having
pyrrolo-quinoline quinone as a coenzyme wherein aspartate 448
(aspartate 424 of SEQ ID NO: 1) in the water-soluble PQQGDH derived
from Acinetobacter calcoaceticus or an amino acid residue
corresponding to said residue is replaced by another amino acid
residue.
9. An isolated modified glucose dehydrogenase having
pyrrolo-quinoline quinone as a coenzyme wherein at least one amino
acid residue is replaced by another amino acid residue in the
region corresponding to residues 268-289 (244-265 of SEQ ID NO: 1)
or 448-468 (424-434 of SEQ ID NO: 1) in the water-soluble PQQGDH
derived from Acinetobacter calcoaceticus.
10. An isolated modified water-soluble glucose dehydrogenase having
pyrrolo-quinoline quinone as a coenzyme wherein glutamate 277
(glutamate 253 of SEQ ID NO: 1) in the water-soluble PQQGDH derived
from Acinetobacter calcoaceticus or an amino acid residue
corresponding to said residue is replaced by another amino acid
residue.
11. An isolated modified water-soluble glucose dehydrogenase having
pyrrolo-quinoline quinone as a coenzyrne wherein isoleucine 278
(isoleucine 254 of SEQ ID NO: 1) in the water-soluble PQQGDH
derived from Acinetobacter calcoaceticus or an amino acid residue
corresponding to said residue is replaced by another amino acid
residue.
12. An isolated modified water-soluble glucose dehydrogenase having
pyrrolo-quinoline quinone as a coenzyme wherein at least one amino
acid residue is replaced by another amino acid residue in the
region defined by residues 268-289 (244-265 of SEQ ID NO: 1) or
448-468 (424-434 of SEQ ID NO: 1) in the amino acid sequence shown
as SEQ ID NO: 1.
13. An isolated modified PQQ glucose dehydrogenase comprising the
sequence (SEQ ID NO: 14): Xaa8 Thr Ala Gly Xaa1 Val Gln Xaa2 Xaa3
Xaa4 Gly Ser Val Thr Xaa5 Thr Leu Glu Asn Pro Gly wherein Xaa1,
Xaa2, Xaa3, Xaa4, Xaa5 and Xaa8 represent any natural amino acid
residue, provided that when Xaa1 represents Asn, Xaa2 represents
Lys, Xaa3 represents Asp, Xaa4 represents Asp and Xaa5 represents
Asn, then Xaa8 does not represent Asp.
14. An isolated modified PQQ glucose dehydrogenase comprising the
sequence (SEQ ID NO: 3): Ser Glu Gln Gly Pro Asn Ser Asp Asp Xaa6
Xaa7 Asn Leu Ile Val Lys Gly Gly Asn TyrGlyTrp wherein Xaa6 and
Xaa7 represent any natural amino acid residue, provided that when
Xaa6 represents Glu, Xaa7 does not represent Ile.
15. The isolated modified glucose dehydrogenase of claim 14 wherein
glutamate 277 (glutamate 253 of SEQ ID NO: 1) is replaced by
another amino acid residue.
16. The isolated modified glucose dehydrogenase of claim 14 wherein
isoleucine 278 (isoleucine 254 of SEQ ID NO: 1) is replaced by
another amino acid residue.
17. A gene encoding the modified glucose dehydrogenase of claim
1.
18. A vector comprising the gene of claim 17.
19. A transformant comprising the gene of claim 17.
20. The transform ant of claim 19 wherein the gene of claim 17 is
integrated into the main chromosome.
21. A glucose assay kit comprising the modified glucose
dehydrogenase of claim 1.
22. A glucose sensor comprising the modified glucose dehydrogenase
of claim
Description
TECHNICAL FIELD
[0001] The present invention relates to the preparation of glucose
dehydrogenases having pyrrolo-quinoline quinone as a coenzyme
(PQQGDH) and their use for glucose assays.
BACKGROUND ART
[0002] Blood glucose is an important marker for diabetes. In the
fermentative production using microorganisms, glucose levels are
assayed for monitoring the process. Conventional glucose assays
were based on enzymatic methods using a glucose oxidase (GOD) or
glucose-6-phosphate dehydrogenase (G6PDH). However, GOD-based
assays required addition of a catalase or peroxidase to the assay
system in order to quantitate the hydrogen peroxide generated by
glucose oxidation reaction. G6PDHs have been used for
spectrophotometric glucose assays, in which case a coenzyme NAD(P)
had to be added to the reaction system.
[0003] Accordingly, an object of the present invention is to
provide a modified water-soluble PQQGDH with improved affinity for
glucose. Another object of the present invention is to provide a
modified water-soluble PQQGDH with high selectivity for glucose in
order to increase the sensitivity for measuring blood glucose
levels.
DISCLOSURE OF THE INVENTION
[0004] We found that PQQGDHs with high affinity for glucose are
useful as novel enzymes alternative to the enzymes that have been
used for enzymatic glucose assays.
[0005] PQQGDHs are glucose dehydrogenases having pyrroloquinoline
quinone as a coenzyme, which catalyze the reaction in which glucose
is oxidized to produce gluconolactone.
[0006] PQQGDHs are known to include membrane-bound enzymes and
water-soluble enzymes. Membrane-bound PQQGDHs are single peptide
proteins having a molecular weight of about 87 kDa and widely found
in various gram-negative bacteria. For example, see A M.
Cleton-Jansen et al., J. Bacteriol. (1990) 172, 6308-6315. On the
other hand, water-soluble PQQGDHs have been identified in several
strains of Acinetobacter calcoaceticus (Biosci. Biotech. Biochem.
(1995), 59(8), 1548-1555), and their structural genes were cloned
to show the amino acid sequences (Mol. Gen. Genet. (1989),
217:430-436). The water-soluble PQQGDH derived from A.
calcoaceticus is a homodimer having a molecular weight of about 50
kDa. It has little homology in primary structure of protein with
other PQQ enzymes.
[0007] Recently, the results of an X-ray crystal structure analysis
of this enzyme were reported to show the higher-order structure of
the enzyme including the active center (J. Mol. Biol., 289, 319-333
(1999), The crystal structure of the apo form of the soluble
quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus
reveals a novel internal conserved sequence repeat; A. Oubrie et
al., The EMBO Journal, 18(19) 5187-5194 (1999), Structure and
mechanism of soluble quinoprotein glucose dehydrogenase, A. Oubrie
et al., PNAS, 96(21), 11787-11791 (1999), Active-site structure of
the soluble quinoprotein glucose dehydrogenase complexed with
methylhydrazine; A covalent cofactor-inhibitor complex, A. Oubrie
et al.). These papers showed that the water-soluble PQQGDH is a
.beta.-propeller protein composed of six W-motifs.
[0008] As a result of careful studies to develop a modified PQQGDH
that can be applied to clinical tests or food analyses by improving
the conventional water-soluble PQQGDH to increase the affinity for
glucose, we succeeded in obtaining an enzyme with high affinity for
glucose by introducing an amino acid change into a specific region
of the water-soluble PQQGDH.
[0009] Accordingly, the present invention provides a modified
water-soluble glucose dehydrogenase having pyrroloquinoline quinone
as a coenzyme characterized in that at least one amino acid residue
in a natural water-soluble glucose dehydrogenase is replaced by
another amino acid residue and it has improved affinity for glucose
as compared with the natural water-soluble glucose dehydrogenase.
The modified PQQGDH of the present invention has a Km value for
glucose lower than the Km value of the natural PQQGDH, preferably
less than 20 mM, more preferably less than 10 mM.
[0010] Preferably, the modified glucose dehydrogenase of the
present invention has increased affinity for glucose though its
affinities for other sugars are unchanged or decreased, whereby it
has higher selectivity for glucose than the natural water-soluble
glucose dehydrogenase. Especially, the reactivity against lactose
or maltose is decreased from that of the wild-type in contrast to
the reactivity to glucose. When the reactivity against glucose is
supposed to be 100%, the activity to lactose or maltose is
preferably 60% or less, more preferably 50% or less, still more
preferably 40% or less.
[0011] In an embodiment of the PQQ glucose dehydrogenase of the
present invention, at least one amino acid residue in the region
corresponding to residues 268-289 or 448-468 in the water-soluble
PQQGDH derived from Acinetobacter calcoaceticus is replaced by
another amino acid residue, i.e. an amino acid residue other than
the relevant amino acid residue in the natural PQQ glucose
dehydrogenase. The amino acid numbering herein starts from the
initiator methionine as the +1 position.
[0012] The term "correspond to" used herein with reference to amino
acid residues or regions means that some amino acid residues or
regions have an equivalent function in two or more structurally
similar but distinct proteins. For example, any region in
water-soluble PQQGDHs derived from other organisms than
Acinetobacter calcoaceticus is said to "correspond to the region
defined by residues 268-289 in the water-soluble PQQGDH derived
from Acinetobacter calcoaceticus" if this region has a high
similarity in the amino acid sequence to the region defined by
residues 268-289 in the water-soluble PQQGDH derived from
Acinetobacter calcoaceticus and this region is reasonably
considered from the secondary structure of the protein to have the
same function in that protein. In addition, the 10th amino acid
residue in this region is said to "correspond to the 277th residue
in the water-soluble PQQGDH derived from Acinetobacter
calcoaceticus".
[0013] In preferred modified PQQGDHs of the present invention, at
least one amino acid residue corresponding to glutamate 277,
isoleucine 278, asparagine 462, asparagine 452, lysine 455,
aspartate 456, aspartate 457 or aspartate 448 in the amino acid
sequence shown as SEQ ID NO: 1 is replaced by another amino acid
residue.
[0014] In more preferred modified PQQGDHs of the present invention,
glutamate 277 is replaced by an amino acid residue selected from
the group consisting of alanine, asparagine, lysine, aspartate,
histidine, glutamine, valine and glycine, or isoleucine 278 is
replaced by phenylalanine in the amino acid sequence shown as SEQ
ID NO: 1.
[0015] In another aspect, modified PQQGDHs of the present invention
comprise the sequence: [0016] Xaa8 Thr Ala Gly Xaa1 Val Gln Xaa2
Xaa3 Xaa4 Gly Ser Val Thr Xaa5 Thr Leu Glu Asn Pro Gly wherein
Xaa1, Xaa2, Xaa3, Xaa4, Xaa5 and Xaa8 represent any natural amino
acid residue, provided that when Xaa1 represents Asn, Xaa2
represents Lys, Xaa3 represents Asp, Xaa4 represents Asp and Xaa5
represents Asn, then Xaa8 does not represent Asp.
[0017] In another aspect, modified PQQGDHs of the present invention
comprise the sequence: [0018] Ser Glu Gln Gly Pro Asn Ser Asp Asp
Xaa6 Xaa7 Asn Leu Ile Val Lys Gly Gly Asn Tyr Gly Trp wherein Xaa6
and Xaa7 represent any natural amino acid residue, provided that
when Xaa6 represents Glu, Xaa7 does not represent Ile.
[0019] The present invention also provides a gene encoding any of
the modified glucose dehydrogenases described above, a vector
containing said gene and a transformant containing said gene, as
well as a glucose assay kit and a glucose sensor comprising a
modified glucose dehydrogenase of the present invention.
[0020] Enzyme proteins of modified PQQGDHs of the present invention
have high affinity for glucose and high oxidation activity for
glucose so that they can be applied to highly sensitive and highly
selective glucose assays.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 shows the structure of the plasmid pGB2 used in the
present invention.
[0022] FIG. 2 shows a scheme for preparing a mutant gene encoding a
modified enzyme of the present invention.
[0023] FIG. 3 shows a glucose assay using a modified PQQGDH of the
present invention.
THE MOST PREFERRED EMBODIMENTS OF THE INVENTION
Structure of Modified POOGDHs
[0024] We introduced random mutations into the coding region of the
gene encoding the water-soluble PQQGDH by error-prone PCR to
construct a library of water-soluble PQQGDHs carrying amino acid
changes. These genes were transformed into E. Coli and screened for
the activity of the PQQGDHs against glucose to give a number of
clones that express PQQGDHs having comparable activities for 20 mM
glucose and 100 mM glucose and improved reactivity against
low-level glucose as compared with that of the wild-type
enzyme.
[0025] Analysis of the nucleotide sequence of one of these clones
showed that Glu 277 had been changed to Gly. When this amino acid
residue was replaced by various other amino acid residues,
excellent mutant enzymes with improved affinity for glucose as
compared with that of the wild type water-soluble PQQGDH were
obtained in every case.
[0026] Then, site-specific mutations were introduced into other
residues near the 277th residue and the affinity for glucose was
determined. Modified enzymes carrying Ile278Phe and Asn279His in
the region defiend by residues 268-289 were prepared and assayed
for the activity to show that these modified enzymes had high
affinity for glucose.
[0027] A number of clones obtained as above were further screened
for clones that express PQQGDHs having activity for 20 mM glucose
comparable to that of the wild-type PQQGDH but activity for 20 mM
lactose lower than that of the wild-type PQQGDH.
[0028] Analysis of the nucleotide sequence of one of these clones
showed that Asn 452 had been changed to Asp. When this residue was
replaced by threonine, lysine, isoleucine, histidine or aspartate,
excellent mutant enzymes with improved selectivity for glucose as
compared with that of the wild type water-soluble PQQGDH were
obtained in every case. Mutations were also introduced into other
residues near the 452nd residue in the same manner. Mutant enzymes
carrying Lys455Ile, Asp456Asn, Asp457Asn, Asn462Asp, Asp448Asn were
constructed. As a result, all the mutant enzymes were found to have
improved selectivity for glucose as shown in Table 4.
[0029] In preferred PQQ glucose dehydrogenases of the present
invention, at least one amino acid residue is replaced by another
amino acid residue in the region corresponding to residues 448-468
in the water-soluble PQQGDH derived from Acinetobacter
calcoaceticus. In preferred modified PQQGDHs of the present
invention, at least one amino acid residue corresponding to
asparagine 462, lysine 452, aspartate 456, aspartate 457 or
aspartate 448 in the amino acid sequence shown as SEQ ID NO: 1 is
replaced by another amino acid residue.
[0030] In another aspect, modified PQQGDHs of the present invention
comprise the sequence: [0031] Xaa8 Thr Ala Gly Xaa1 Val Gln Xaa2
Xaa3 Xaa4 Gly Ser Val Thr Xaa5 Thr Leu Glu Asn Pro Gly wherein
Xaa1, Xaa2, Xaa3, Xaa4, Xaa5 and Xaa8 represent any natural amino
acid residue, provided that when Xaa1 represents Asn, Xaa2
represents Lys, Xaa3 represents Asp, Xaa4 represents Asp and Xaa5
represents Asn, then Xaa8 does not represent Asp.
[0032] In other preferred PQQ glucose dehydrogenases of the present
invention, at least one amino acid residue is replaced by another
amino acid residue in the region corresponding to residues 268-289
in the amino acid sequence shown as SEQ ID NO: 1. In especially
preferred modified PQQGDHs of the present invention, glutamate 277
is replaced by an amino acid residue selected from the group
consisting of alanine, asparagine, lysine, aspartate, histidine,
glutamine, valine and glycine, or isoleucine 278 is replaced by
phenylalanine in the amino acid sequence shown as SEQ ID NO: 1.
[0033] In another aspect, modified PQQGDHs of the present invention
comprise the sequence: [0034] Ser Glu Gln Gly Pro Asn Ser Asp Asp
Xaa6 Xaa7 Asn Leu Ile Val Lys Gly Gly Asn Tyr Gly Trp wherein Xaa6
and Xaa7 represent any natural amino acid residue, provided that
when Xaa6 represents Glu, Xaa7 does not represent Ile.
[0035] In modified glucose dehydrogenases of the present invention,
other amino acid residues may be partially deleted or substituted
or other amino acid residues may be added so far as glucose
dehydrogenase activity is retained.
[0036] Those skilled in the art can also replace an amino acid
residue in water-soluble PQQGDHs derived from other bacteria
according to the teaching herein to obtain modified glucose
dehydrogenases with improved affinity for glucose. Particularly,
amino acid residues corresponding to glutamate 277, isoleucine 278,
asparagine 462, lysine 452, aspartate 455, aspartate 456, aspartate
457 and aspartate 448 in the water-soluble PQQGDH derived from
Acinetobacter calcoaceticus can be readily identified by comparing
the primary structures of proteins in alignment or comparing the
secondary structures predicted from the primary structures of the
enzymes. Modified glucose dehydrogenases with improved affinity for
substrate can be obtained by replacing such amino acid residues
according to the present invention. These modified glucose
dehydrogenases are also within the scope of the present
invention.
Process for Preparing Modified PQQGDHs
[0037] The sequence of the gene encoding the wild-type
water-soluble PQQGDH derived from Acinetobacter calcoaceticus is
defined by SEQ ID NO: 2.
[0038] Genes encoding modified PQQGDHs of the present invention can
be constructed by replacing the nucleotide sequence encoding a
specific amino acid residue in the gene encoding the wild-type
water-soluble PQQGDH by the nucleotide sequence encoding an amino
acid residue to be substituted. Various techniques for such
site-specific nucleotide sequence substitution are known in the art
as described in Sambrook et al., "Molecular Cloning: A Laboratory
Manual", Second Edition, 1989, Cold Spring Harbor Laboratory Press,
New York, for example.
[0039] Thus obtained mutant gene is inserted into a gene expression
vector (for example, a plasmid) and transformed into an appropriate
host (for example, E. coli). A number of vector/host systems for
expressing a foreign protein are known and various hosts such as
bacteria, yeasts or cultured cells are suitable.
[0040] Random mutations are introduced by error-prone PCR into a
target region to construct a gene library of modified water-soluble
PQQGDHs carrying mutations in the target region. These genes are
transformed into E. coli to screen each clone for the affinity of
the PQQGDH for glucose. Water-soluble PQQGDHs are secreted into the
periplasmic space when they are expressed in E. coli, so that they
can be easily assayed for enzyme activity using the E. coli cells.
This library is combined with a PMS-DCIP dye in the presence of 20
mM glucose to visually determine the PQQGDH activity so that clones
showing activity comparable to the activity for 100 mM glucose are
selected and analyzed for the nucleotide sequence to confirm the
mutation.
[0041] In order to obtain modified PQQGDHs with improved
selectivity for glucose, this library is combined with a PMS-DCIP
dye to visually determine the PQQGDH activity so that clones
showing activity for 20 mM glucose comparable to that of the
wild-type PQQGDH but activity for 20 mM lactose lower than that of
the wild-type PQQGDH are selected and analyzed for the nucleotide
sequence to confirm the mutation.
[0042] Thus obtained transformed cells expressing modified PQQGDHs
are cultured and harvested by centrifugation or other means from
the culture medium, and then disrupted with a French press or
osmotically shocked to release the periplasmic enzyme into the
medium. The enzyme may be ultracentrifuged to give a water-soluble
PQQGDH-containing fraction. Alternatively, the expressed PQQGDH may
be secreted into the medium by using an appropriate host/vector
system. The resulting water-soluble fraction is purified by ion
exchange chromatography, affinity chromatography, HPLC and the like
to prepare a modified PQQGDH of the present invention.
Method for Assaying Enzyme Activity
[0043] PQQGDHs of the present invention associate with PQQ as a
coenzyme in catalyzing the reaction in which glucose is oxidized to
produce gluconolactone.
[0044] The enzyme activity can be assayed by using the
color-developing reaction of a redox dye to measure the amount of
PQQ reduced with PQQGDH-catalyzed oxidation of glucose. Suitable
color-developing reagents include PMS (phenazine methosulfate)-DCIP
(2,6-dichlorophenolindophenol), potassium ferricyanide and
ferrocene, for example.
Affinity for Glucose
[0045] Modified PQQGDHs of the present invention have greatly
improved affinity for glucose as compared with that of the wild
type. Thus, modified PQQGDHs have a Km value for glucose that is
greatly lower than the Km value for glucose of the wild-type
PQQGDH. Among modified PQQGDHs, the Glu277Lys variant has a Km
value for glucose of 8.8 mM and a maximum activity comparable to
that of the wild-type enzyme so that it has improved reactivity
against glucose at low levels.
[0046] Therefore, assay kits or enzyme sensors prepared with
modified enzymes of the present invention have the excellent
advantages that they can detect glucose at low levels because of
the high sensitivity for glucose assays.
Evaluation Method of Selectivity
[0047] Selectivity for glucose of PQQGDHs of the present invention
can be evaluated by assaying the enzyme activity as described above
using various sugars such as 2-deoxy-D-glucose, mannose, allose,
3-o-methyl-D-glucose, galactose, xylose, lactose and maltose as
substrates and determining the relative activity to the activity
for glucose.
Glucose Assay Kit
[0048] The present invention also relates to a glucose assay kit
comprising a modified PQQGDH according to the present invention.
The glucose assay kit of the present invention comprises a modified
PQQGDH according to the present invention in an amount enough for
at least one run of assay. In addition to the modified PQQGDH
according to the present invention, the kit typically comprises a
necessary buffer for the assay, a mediator, standard glucose
solutions for preparing a calibration curve and instructions.
Modified PQQGDHs according to the present invention can be provided
in various forms such as freeze-dried reagents or solutions in
appropriate preservative solutions. Modified PQQGDHs according to
the present invention are preferably provided in the form of a
holoenzyme, though they may also be provided as an apoenzyme and
converted into a holoenzyme before use.
Glucose Sensor
[0049] The present invention also relates to a glucose sensor using
a modified PQQGDH according to the present invention. Suitable
electrodes include carbon, gold, platinum and the like electrodes,
on which an enzyme of the present invention is immobilized by using
a crosslinking agent; encapsulation in a polymer matrix; coating
with a dialysis membrane; using a photo-crosslinkable polymer, an
electrically conductive polymer or a redox polymer; fixing the
enzyme in a polymer or adsorbing it onto the electrode with an
electron mediator including ferrocene or its derivatives; or any
combination thereof. Modified PQQGDHs of the present invention are
preferably immobilized in the form of a holoenzyme on an electrode,
though they may be immobilized as an apoenzyme and PQQ may be
provided as a separate layer or in a solution. Typically, modified
PQQGDHs of the present invention are immobilized on a carbon
electrode with glutaraldehyde and then treated with an
amine-containing reagent to block glutaraldehyde.
[0050] Glucose levels can be measured as follows. PQQ, CaCl.sub.2
and a mediator are added to a thermostat cell containing a buffer
and kept at a constant temperature. Suitable mediators include, for
example, potassium ferricyanide and phenazine methosulfate. An
electrode on which a modified PQQGDH of the present invention has
been immobilized is used as a working electrode in combination with
a counter electrode (e.g. a platinum electrode) and a reference
electrode (e.g. an Ag/AgCl electrode). After a constant voltage is
applied to the carbon electrode to reach a steady current, a
glucose-containing sample is added to measure the increase in
current. The glucose level in the sample can be calculated from a
calibration curve prepared with glucose solutions at standard
concentrations.
[0051] The disclosures of all the patents and documents cited
herein are entirely incorporated herein as reference. The present
application claims priority based on Japanese Patent Applications
Nos. 1999-124285 and 2000-9137, the disclosure of which is entirely
incorporated herein as reference.
[0052] The following examples further illustrate the present
invention without, however, limiting the same thereto.
EXAMPLE 1
Construction and Screening of a Mutant PQQGDH Gene Library:
[0053] The plasmid pGB2 was obtained by inserting the structural
gene encoding the PQQGDH derived from Acinetobacter calcoaceticus
into the multicloning site of the vector pTrc99A (Pharmacia) (FIG.
1). This plasmid was used as a template to introduce random
mutations into various regions by error-prone PCR. The PCR reaction
was carried out in a solution having the composition shown in Table
1 under the conditions of 94.degree. C. for 3 minutes, 30 cycles of
94.degree. C. for 3 minutes, 50.degree. C. for 2 minutes and
72.degree. C. for 2 minutes, and finally 72.degree. C. for 10
minutes. TABLE-US-00001 TABLE 1 TaqDNA polymerase (5 U/.mu.l) 0.5
.mu.l Template DNA 1.0 .mu.l Forward primer ABF 4.0 .mu.l Reverse
primer ABR 4.0 .mu.l 10.times. Taq polymerase buffer 10.0 .mu.l 1 M
.beta.-mercaptoethanol 1.0 .mu.l DMSO 10.0 .mu.l 5 mM MnCl.sub.2
10.0 .mu.l 10 mM dGTP 2.0 .mu.l 2 mM dATP 2.0 .mu.l 10 mM dCTP 2.0
.mu.l 10 mM dTTP 2.0 .mu.l H.sub.2O 51.5 .mu.l 100.0 .mu.l
[0054] The resulting mutant water-soluble PQQGDH library was
transformed into E. coli and each colony formed was transferred to
a microtiter plate. The colony was further replica-plated on a
first plate containing 10 mM glucose and PMS-DCIP and a second
plate containing 100 mM glucose and PMS-CDIP, and both were
visually evaluated for the PQQGDH activity. A number of clones
showing comparable PQQGDH activities in both plates were
obtained.
[0055] One of these clones was randomly selected and analyzed for
the nucleotide sequence to show that glutamate 277 had been changed
to glycine.
EXAMPLE 2
[0056] Each colony obtained in Example 1 was transferred to a
microtiter plate. The colony was replica-plated on a first plate
containing 20 mM glucose and PMS-DCIP and a second plate containing
20 mM lactose and PMS-CDIP, and both were visually evaluated for
the PQQGDH activity. A number of clones showing a greatly lower
activity for lactose than glucose in both plates were obtained.
[0057] One of these clones was randomly selected and analyzed for
the nucleotide sequence to show that asparagine 452 had been
changed to aspartate.
EXAMPLE 3
Construction of modified PQQGDH genes:
[0058] Based on the structural gene of the PQQGDH derived from
Acinetobacter calcoaceticus shown as SEQ ID NO: 2, the nucleotide
sequence encoding glutamate 277 or isoleucine 278 was replaced by
the nucleotide sequences encoding given amino acid residues by
site-directed mutagenesis according to a standard method as shown
in FIG. 2 using the plasmid pGB2. Table 2 shows the sequences of
the synthetic oligonucleotide target primers used for mutagenesis.
In Table 2, "E277A" means that glutamate 277 is replaced by
aspartate, for example. TABLE-US-00002 TABLE 2 E277A 5'- GAG GTT
AAT TGC ATC GTC AGA G -3' E277N 5'- C AAT GAG GTT AAT GTT ATC GTC
AGA GTT TG -3' E277K 5'- GAG GTT AAT ATC ATC GTC AGA G -3' E277D
5'- GAG GTT AAT TTT ATC GTC AGA G -3' E277H 5'- C AAT GAG GTT AAT
GTG ATC GTC AGA GTT TG -3' E277Q 5'- GAG GTT AAT TTG ATC GTC AGA G
-3' E277V 5'- C AAT GAG GTT AAT TAC ATC GTC AGA GTT TG -3' E277G
5'- GAG GTT AAT TCC ATC GTC AGA G -3' I278F 5'- C AAT GAG GTT GAA
TTC ATC GTC AGA G -3' N279H 5'- GAC AAT GAG GTC AAT TTC ATC GTC AGA
GTT -3'
[0059] A KpnI-HindIII fragment containing a part of the gene
encoding the PQQGDH derived from Acinetobacter calcoaceticus was
integrated into the vector plasmid pKF18k (Takara Shuzo Co., Ltd.)
and used as a template. Fifty fmols of this template, 5 pmol of the
selection primer attached to the Mutan.TM.-Express Km Kit (Takara
Shuzo Co., Ltd.) and 50 pmol of the phosphorylated target primer
were mixed with the annealing buffer attached to the kit in an
amount equivalent to 1/10 of the total volume (20 .mu.l), and the
mixture was heated at 100.degree. C. for 3 minutes to denature the
plasmid into a single strand. The selection primer serves for
reversion of dual amber mutations on the kanamycin-resistance gene
of pKF18k. The mixture was placed on ice for 5 minutes to anneal
the primers. To this mixture were added 3 .mu.l of the extension
buffer attached to the kit, 1 .mu.l of T4 DNA ligase, 1 l of T4 DNA
polymerase and 5 .mu.l of sterilized water to synthesize a
complementary strand.
[0060] The synthetic strand was transformed into a DNA mismatch
repair-deficient strain E. coli BMH71-18mutS and shake-cultured
overnight to amplify the plasmid.
[0061] Then, the plasmid copies were extracted from the cultures
and transformed into E. coli MV1184 and then extracted from the
colonies. These plasmids were sequenced to confirm the introduction
of the intended mutations. These fragments were substituted for the
KpnI-HindIII fragment of the gene encoding the wild-type PQQGDH on
the plasmid pGB2A to construct genes for modified PQQGDHs.
[0062] An oligonucleotide target primer of the sequence: [0063]
5'-C ATC TTT TTG GAC ATG TCC GGC AGT AT-3' was synthesized in the
same manner to substitute histidine for asparagine 452.
Site-directed mutagenesis was performed by the method shown in FIG.
2 using the plasmid pGB2. Genes for modified PQQGDHs carrying
mutations Asp448Asn, Asn452Asp, Asn452His, Asn452Lys, Asn452Thr,
Asn452Ile, Lys455Ile, Asp456Asn, Asp457Asn and Asn462Asp were also
constructed.
EXAMPLE 4
Preparation of Modified Enzymes
[0064] The gene encoding the wild-type or each modified PQQGDH was
inserted into the multicloning site of an E. coli expression vector
pTrc99A (Pharmacia), and the resulting plasmid was transformed into
the E. coli strain DH5.alpha.. The transformant was shake-cultured
at 37.degree. C. overnight on 450 ml of L medium (containing 50
.mu.g/ml of ampicillin) in a Sakaguchi flask, and inoculated on 7 l
of L medium containing 1 mM CaCl.sub.2 and 500 .mu.M PQQ. About 3
hours after starting cultivation, isopropyl thiogalactoside was
added at a final concentration of 0.3 mM, and cultivation was
further continued for 1.5 hours. The cultured cells were harvested
from the medium by centrifugation (5,000.times. g, 10 min,
4.degree. C.), and washed twice with a 0.85% NaCl solution. The
collected cells were disrupted with a French press, and centrifuged
(10,000.times. g, 15 min, 4.degree. C.) to remove undisrupted
cells. The supernatant was ultracentrifuged (160,500.times. g
(40,000 r.p.m.), 90 min, 4.degree. C.) to give a water-soluble
fraction, which was used in the subsequent examples as a crude
enzyme sample.
[0065] Thus obtained water-soluble fraction was further dialyzed
against 10 mM phosphate buffer, pH 7.0 overnight. The dialyzed
sample was adsorbed to a cation chromatographic column TSKgel
CM-TOYOPEARL 650M (Tosoh Corp.), which had been equilibrated with
10 mM phosphate buffer, pH 7.0. This column was washed with 750 ml
of 10 mM phosphate buffer, pH 7.0 and then the enzyme was eluted
with 10 mM phosphate buffer, pH 7.0 containing 0-0.2 M NaCl at a
flow rate of 5 ml/min. Fractions having GDH activity were collected
and dialyzed against 10 mM MOPS-NAOH buffer, pH 7.0 overnight.
Thus, an electrophoretically homogeneous modified PQQGDH protein
was obtained. This was used in the subsequent examples as a
purified enzyme sample.
EXAMPLE 5
Assay of Enzyme Activity
[0066] Enzyme activity was assayed by using PMS (phenazine
methosulfate)-DCIP (2,6-dichlorophenolindophenol) in 10 mM
MOPS-NaOH buffer (pH 7.0) to monitor changes in the absorbance of
DCIP at 600 nm with a spectrophotometer and expressing the reaction
rate of the enzyme as the rate of decrease in the absorbance. The
enzyme activity for reducing 1 .mu.mol of DCIP in 1 minute was 1 U.
The molar extinction coefficient of DCIP at pH 7.0 was 16.3
mM.sup.-1.
EXAMPLE 6
Evaluation of Affinity of Crude Enzyme Samples for Glucose:
[0067] Each of the crude enzyme samples of the wild-type and
modified PQQGDHs obtained in Example 4 was converted into a
holoenzyme in the presence of 1 .mu.M PQQ and 1 mM CaCl.sub.2 for 1
hour or longer. A 187 .mu.l-aliquot was combined with 3 .mu.l of an
activating reagent (prepared from 48 .mu.l of 6 mM DCIP, 8 .mu.l of
600 mM PMS and 16 l of 10 mM phosphate buffer, pH 7.0) and 10 .mu.l
of D-glucose solutions at various concentrations, and assayed for
the enzyme activity at room temperature by the method shown in
Example 5. The Km was determined by plotting the substrate
concentration vs. enzyme activity. The results are shown in Table
3. TABLE-US-00003 TABLE 3 Km (mM) Wild type 26.0 G277A 1.5 G277N
1.2 G277K 8.9 G277D 7.4 G277H 7.7 G277Q 4.3 G277V 2.5 G277G 0.3
I278F 7.0 N279H 15.7 N452T 12.5 N462D 12.2 N462K 11.0 N462Y
20.4
[0068] The Km value of the wild-type PQQGDH for glucose reported to
date was about 25 mM. In contrast, all the enzymes constructed here
to carry mutations in glutamate 277 and Ile278Phe had a Km value
for glucose of less than 10 mM. These results show that modified
PQQGDHs of the present invention have high affinity for
glucose.
EXAMPLE 7
Evaluation of Affinity of Purified Enzyme Samples for Glucose:
[0069] Each of the purified samples of the wild-type enzyme and the
modified enzyme Glu277Lys obtained in Example 4 was converted into
a holoenzyme in the presence of 1 .mu.M PQQ and 1 mM CaCl.sub.2 for
1 hour or longer in the same manner as in Example 6. A 187
.mu.l-aliquot was combined with 3 .mu.l of an activating reagent
(prepared from 48 l of 6 mM DCIP, 8 .mu.l of 600 mM PMS and 16
.mu.l of 10 mM phosphate buffer, pH 7.0) and 10 l of D-glucose
solutions at various concentrations, and assayed for the enzyme
activity at room temperature by the method shown in Example 5. The
Km and Vmax were determined by plotting the substrate concentration
vs. enzyme activity. The Glu277Lys variant had a Km value for
glucose of about 8.8 mM and a Vmax value of 3668 U/mg. The Km value
of the wild-type PQQGDH for glucose reported to date was about 25
mM with the Vmax value being 2500-7000 U/mg depending on the
measurement conditions. These results show that the modified PQQGDH
Glu277Lys is an enzyme having remarkably improved affinity for
glucose and high activity comparable to that of the wild-type
PQQGDH.
EXAMPLE 8
Evaluation of Substrate Specificity:
[0070] Crude samples of various modified enzymes were tested for
substrate specificity. Each of the crude samples of the wild-type
and various modified PQQGDHs was converted into a holoenzyme in the
presence of 1 .mu.M PQQ and 1 mM CaCl.sub.2 for 1 hour or longer. A
187 .mu.l-aliquot was combined with 3 .mu.l of an activating
reagent (containing 6 mM DCIP, 600 mM PMS and 10 mM phosphate
buffer, pH 7.0) and a substrate. The substrates tested were 400 mM
glucose, lactose and maltose at a final concentration of 20 mM, and
each sample was incubated with 10 l of each substrate at room
temperature for 30 minutes and assayed for the enzyme activity in
the same manner as in Example 5 to determine the relative activity
expressed as the percentage of the activity for glucose. As shown
in Table 4, all the modified enzymes of the present invention
showed higher selectivity for glucose than that of the wild-type
enzyme. TABLE-US-00004 TABLE 4 Glucose Lactose Maltose Wild-type
100% 61% 61% Asp448Asn 100% 48% 36% Asn452Asp 100% 56% 50%
Asn452His 100% 39% 39% Asn452Lys 100% 55% 42% Asn452Thr 100% 42%
30% Asn452Ile 100% 36% 28% Lys455Ile 100% 49% 37% Asp456Asn 100%
59% 41% Asp457Asn 100% 43% 32% Asn462Asp 100% 52% 41%
EXAMPLE 9
Glucose Assay:
[0071] Modified PQQGDHs were used for assaying glucose. Each of the
modified enzymes Glu277Lys and Asn452Thr was converted into a
holoenzyme in the presence of 1 .mu.M PQQ and 1 mM CaCl.sub.2 for 1
hour or longer, and assayed for the enzyme activity in the presence
of glucose at various concentrations as well as 5 .mu.M PQQ and 10
mM CaCl.sub.2 by the method described in Example 5 based on changes
of the absorbance of DCIP at 600 nm. As shown in FIG. 3, the
modified PQQGDH Asn452Thr could be used for assaying glucose in the
range of 0.1-20 mM. Similar results were obtained with the modified
PQQGDH Glu277Lys.
EXAMPLE 10
Preparation and Evaluation of an Enzyme Sensor:
[0072] Five units each of the modified enzymes Glu277Lys and
Asn452Thr were freeze-dried with 20 mg of carbon paste. After
thorough mixing, the mixture was applied only on the surface of a
carbon paste electrode preliminarily filled with about 40 mg of
carbon paste and polished on a filter paper. This electrode was
treated in 10 mM MOPS buffer (pH 7.0) containing 1% glutaraldehyde
at room temperature for 30 minutes followed by 10 mM MOPS buffer
(pH 7.0) containing 20 mM lysine at room temperature for 20 minutes
to block glutaraldehyde. The electrode was equilibrated in 10 mM
MOPS. buffer (pH 7.0) at room temperature for 1 hour or longer and
then stored at 4.degree. C.
[0073] Thus prepared enzyme sensor was used to measure glucose
levels. The enzyme sensor having a modified PQQGDH of the present
invention immobilized thereon can be used for assaying glucose in
the range of 0.1 mM-5 mM.
INDUSTRIAL APPLICABILITY
[0074] Modified PQQGDHs of the present invention have high affinity
for glucose so that they are expected to provide the advantages
that assay kits or enzyme sensors prepared with such enzymes can
measure glucose at lower levels with remarkably improved
sensitivity as compared with conventional natural PQQGDHs.
Sequence CWU 1
1
15 1 454 PRT Acinetobacter calcoaceticus 1 Asp Val Pro Leu Thr Pro
Ser Gln Phe Ala Lys Ala Lys Ser Glu Asn 1 5 10 15 Phe Asp Lys Lys
Val Ile Leu Ser Asn Leu Asn Lys Pro His Ala Leu 20 25 30 Leu Trp
Gly Pro Asp Asn Gln Ile Trp Leu Thr Glu Arg Ala Thr Gly 35 40 45
Lys Ile Leu Arg Val Asn Pro Glu Ser Gly Ser Val Lys Thr Val Phe 50
55 60 Gln Val Pro Glu Ile Val Asn Asp Ala Asp Gly Gln Asn Gly Leu
Leu 65 70 75 80 Gly Phe Ala Phe His Pro Asp Phe Lys Asn Asn Pro Tyr
Ile Tyr Ile 85 90 95 Ser Gly Thr Phe Lys Asn Pro Lys Ser Thr Asp
Lys Glu Leu Pro Asn 100 105 110 Gln Thr Ile Ile Arg Arg Tyr Thr Tyr
Asn Lys Ser Thr Asp Thr Leu 115 120 125 Glu Lys Pro Val Asp Leu Leu
Ala Gly Leu Pro Ser Ser Lys Asp His 130 135 140 Gln Ser Gly Arg Leu
Val Ile Gly Pro Asp Gln Lys Ile Tyr Tyr Thr 145 150 155 160 Ile Gly
Asp Gln Gly Arg Asn Gln Leu Ala Tyr Leu Phe Leu Pro Asn 165 170 175
Gln Ala Gln His Thr Pro Thr Gln Gln Glu Leu Asn Gly Lys Asp Tyr 180
185 190 His Thr Tyr Met Gly Lys Val Leu Arg Leu Asn Leu Asp Gly Ser
Ile 195 200 205 Pro Lys Asp Asn Pro Ser Phe Asn Gly Val Val Ser His
Ile Tyr Thr 210 215 220 Leu Gly His Arg Asn Pro Gln Gly Leu Ala Phe
Thr Pro Asn Gly Lys 225 230 235 240 Leu Leu Gln Ser Glu Gln Gly Pro
Asn Ser Asp Asp Glu Ile Asn Leu 245 250 255 Ile Val Lys Gly Gly Asn
Tyr Gly Trp Pro Asn Val Ala Gly Tyr Lys 260 265 270 Asp Asp Ser Gly
Tyr Ala Tyr Ala Asn Tyr Ser Ala Ala Ala Asn Lys 275 280 285 Ser Ile
Lys Asp Leu Ala Gln Asn Gly Val Lys Val Ala Ala Gly Val 290 295 300
Pro Val Thr Lys Glu Ser Glu Trp Thr Gly Lys Asn Phe Val Pro Pro 305
310 315 320 Leu Lys Thr Leu Tyr Thr Val Gln Asp Thr Tyr Asn Tyr Asn
Asp Pro 325 330 335 Thr Cys Gly Glu Met Thr Tyr Ile Cys Trp Pro Thr
Val Ala Pro Ser 340 345 350 Ser Ala Tyr Val Tyr Lys Gly Gly Lys Lys
Ala Ile Thr Gly Trp Glu 355 360 365 Asn Thr Leu Leu Val Pro Ser Leu
Lys Arg Gly Val Ile Phe Arg Ile 370 375 380 Lys Leu Asp Pro Thr Tyr
Ser Thr Thr Tyr Asp Asp Ala Val Pro Met 385 390 395 400 Phe Lys Ser
Asn Asn Arg Tyr Arg Asp Val Ile Ala Ser Pro Asp Gly 405 410 415 Asn
Val Leu Tyr Val Leu Thr Asp Thr Ala Gly Asn Val Gln Lys Asp 420 425
430 Asp Gly Ser Val Thr Asn Thr Leu Glu Asn Pro Gly Ser Leu Ile Lys
435 440 445 Phe Thr Tyr Lys Ala Lys 450 2 1612 DNA Acinetobacter
calcoaceticus 2 agctactttt atgcaacaga gcctttcaga aatttagatt
ttaatagatt cgttattcat 60 cataatacaa atcatataga gaactcgtac
aaacccttta ttagaggttt aaaaattctc 120 ggaaaatttt gacaatttat
aaggtggaca catgaataaa catttattgg ctaaaattgc 180 tttattaagc
gctgttcagc tagttacact ctcagcattt gctgatgttc ctctaactcc 240
atctcaattt gctaaagcga aatcagagaa ctttgacaag aaagttattc tatctaatct
300 aaataagccg catgctttgt tatggggacc agataatcaa atttggttaa
ctgagcgagc 360 aacaggtaag attctaagag ttaatccaga gtcgggtagt
gtaaaaacag tttttcaggt 420 accagagatt gtcaatgatg ctgatgggca
gaatggttta ttaggttttg ccttccatcc 480 tgattttaaa aataatcctt
atatctatat ttcaggtaca tttaaaaatc cgaaatctac 540 agataaagaa
ttaccgaacc aaacgattat tcgtcgttat acctataata aatcaacaga 600
tacgctcgag aagccagtcg atttattagc aggattacct tcatcaaaag accatcagtc
660 aggtcgtctt gtcattgggc cagatcaaaa gatttattat acgattggtg
accaagggcg 720 taaccagctt gcttatttgt tcttgccaaa tcaagcacaa
catacgccaa ctcaacaaga 780 actgaatggt aaagactatc acacctatat
gggtaaagta ctacgcttaa atcttgatgg 840 aagtattcca aaggataatc
caagttttaa cggggtggtt agccatattt atacacttgg 900 acatcgtaat
ccgcagggct tagcattcac tccaaatggt aaattattgc agtctgaaca 960
aggcccaaac tctgacgatg aaattaacct cattgtcaaa ggtggcaatt atggttggcc
1020 gaatgtagca ggttataaag atgatagtgg ctatgcttat gcaaattatt
cagcagcagc 1080 caataagtca attaaggatt tagctcaaaa tggagtaaaa
gtagccgcag gggtccctgt 1140 gacgaaagaa tctgaatgga ctggtaaaaa
ctttgtccca ccattaaaaa ctttatatac 1200 cgttcaagat acctacaact
ataacgatcc aacttgtgga gagatgacct acatttgctg 1260 gccaacagtt
gcaccgtcat ctgcctatgt ctataagggc ggtaaaaaag caattactgg 1320
ttgggaaaat acattattgg ttccatcttt aaaacgtggt gtcattttcc gtattaagtt
1380 agatccaact tatagcacta cttatgatga cgctgtaccg atgtttaaga
gcaacaaccg 1440 ttatcgtgat gtgattgcaa gtccagatgg gaatgtctta
tatgtattaa ctgatactgc 1500 cggaaatgtc caaaaagatg atggctcagt
aacaaataca ttagaaaacc caggatctct 1560 cattaagttc acctataagg
ctaagtaata cagtcgcatt aaaaaaccga tc 1612 3 22 PRT Acinetobacter
calcoaceticus MISC_FEATURE (10)..(10) Xaa is any natural amino acid
residue 3 Ser Glu Gln Gly Pro Asn Ser Asp Asp Xaa Xaa Asn Leu Ile
Val Lys 1 5 10 15 Gly Gly Asn Tyr Gly Trp 20 4 22 DNA Artificial
Sequence synthetic oligonucleotide target primer used for
mutagenesis of DNA derived from Acinetobacter calcoaceticus 4
gaggttaatt gcatcgtcag ag 22 5 30 DNA Artificial Sequence synthetic
oligonucleotide target primer used for mutagenesis of DNA derived
from Acinetobacter calcoaceticus 5 caatgaggtt aatgttatcg tcagagtttg
30 6 22 DNA Artificial Sequence synthetic oligonucleotide target
primer used for mutagenesis of DNA derived from Acinetobacter
calcoaceticus 6 gaggttaata tcatcgtcag ag 22 7 22 DNA Artificial
Sequence synthetic oligonucleotide target primer used for
mutagenesis of DNA derived from Acinetobacter calcoaceticus 7
gaggttaatt ttatcgtcag ag 22 8 30 DNA Artificial Sequence synthetic
oligonucleotide target primer used for mutagenesis of DNA derived
from Acinetobacter calcoaceticus 8 caatgaggtt aatgtgatcg tcagagtttg
30 9 22 DNA Artificial Sequence synthetic oligonucleotide target
primer used for mutagenesis of DNA derived from Acinetobacter
calcoaceticus 9 gaggttaatt tgatcgtcag ag 22 10 30 DNA Artificial
Sequence synthetic oligonucleotide target primer used for
mutagenesis of DNA derived from Acinetobacter calcoaceticus 10
caatgaggtt aattacatcg tcagagtttg 30 11 22 DNA Artificial Sequence
synthetic oligonucleotide target primer used for mutagenesis of DNA
derived from Acinetobacter calcoaceticus 11 gaggttaatt ccatcgtcag
ag 22 12 26 DNA Artificial Sequence synthetic oligonucleotide
target primer used for mutagenesis of DNA derived from
Acinetobacter calcoaceticus 12 caatgaggtt gaattcatcg tcagag 26 13
30 DNA Artificial Sequence synthetic oligonucleotide target primer
used for mutagenesis of DNA derived from Acinetobacter
calcoaceticus 13 gacaatgagg tgaatttcat cgtcagagtt 30 14 21 PRT
Acinetobacter calcoaceticus MISC_FEATURE (1)..(1) Xaa is any
natural amino acid; however, it is not Asp when Xaa at residue 5 is
Asn, Xaa at residue 8 is Lys, Xaa at residue 9 is Asp, Xaa at
residue 10 is Asp, and Xaa at residue 15 is Asn 14 Xaa Thr Ala Gly
Xaa Val Gln Xaa Xaa Xaa Gly Ser Val Thr Xaa Thr 1 5 10 15 Leu Glu
Asn Pro Gly 20 15 27 DNA Artificial Sequence synthetic
oligonucleotide target primer used for mutagenesis of DNA derived
from Acinetobacter calcoaceticus 15 catctttttg gacatgtccg gcagtat
27
* * * * *