U.S. patent application number 11/313684 was filed with the patent office on 2006-05-11 for glucose dehydrogenases.
Invention is credited to Koji Sode.
Application Number | 20060099698 11/313684 |
Document ID | / |
Family ID | 26442061 |
Filed Date | 2006-05-11 |
United States Patent
Application |
20060099698 |
Kind Code |
A1 |
Sode; Koji |
May 11, 2006 |
Glucose dehydrogenases
Abstract
Modified water-soluble glucose dehydrogenase 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 thermal stability.
Inventors: |
Sode; Koji; (Tokyo,
JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
26442061 |
Appl. No.: |
11/313684 |
Filed: |
December 22, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09958231 |
Oct 5, 2001 |
|
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PCT/JP00/02322 |
Apr 10, 2000 |
|
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11313684 |
Dec 22, 2005 |
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Current U.S.
Class: |
435/189 ;
435/252.3; 435/320.1; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12Q 1/34 20130101; C12Q
1/006 20130101; C12Y 101/05002 20130101; C12Y 101/03004 20130101;
C12N 9/0006 20130101 |
Class at
Publication: |
435/189 ;
435/069.1; 435/252.3; 435/320.1; 536/023.2 |
International
Class: |
C12N 9/02 20060101
C12N009/02; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C12N 15/74 20060101 C12N015/74; C12N 1/21 20060101
C12N001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 8, 1999 |
JP |
101143/1999 |
Jan 18, 2000 |
JP |
9152/2000 |
Claims
1. A modified glucose dehydrogenase having pyrrolo-quinoline
quinone as a coenzyme wherein an amino acid residue corresponding
to serine 231 in the water-soluble PQQGDH derived from
Acinetobacter calcoaceticus is replaced by another amino acid
residue.
2. A modified glucose dehydrogenase having pyrrolo-quinoline
quinone as a coenzyme wherein an amino acid residue corresponding
to glutamine 209 in the water-soluble PQQGDH derived from
Acinetobacter calcoaceticus is replaced by another amino acid
residue.
3. A modified glucose dehydrogenase having pyrrolo-quinoline
quinone as a coenzyme wherein an amino acid residue corresponding
to glutamate 210 in the water-soluble PQQGDH derived from
Acinetobacter calcoaceticus is replaced by another amino acid
residue.
4. A modified glucose dehydrogenase having pyrrolo-quinoline
quinone as a coenzyme wherein an amino acid residue corresponding
to aspartate 420 in the water-soluble PQQGDH derived from
Acinetobacter calcoaceticus is replaced by another amino acid
residue.
5. A modified glucose dehydrogenase having pyrrolo-quinoline
quinone as a coenzyme wherein an amino acid residue corresponding
to alanine 421 in the water-soluble PQQGDH derived from
Acinetobacter calcoaceticus is replaced by another amino acid
residue.
6. A 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 one or more regions
selected from the group consisting of the regions defined by
residues 48-53, 60-62, 69-71, 79-82, 91-101, 110-115, 127-135,
147-150, 161-169, 177-179, 186-221, 227-244, 250-255, 261-263,
271-275, 282-343, 349-377, 382-393, 400-403, 412-421, 427-432,
438-441 and 449-468 in the amino acid sequence shown as SEQ ID NO:
1, characterized in that it has higher thermal stability than that
of the water-soluble glucose dehydrogenase derived from
Acinetobacter calcoaceticus.
7. The modified glucose dehydrogenase of claim 3 wherein at least
one amino acid residue is replaced by another amino acid residue in
the region defined by residues 227-244 in the amino acid sequence
shown as SEQ ID NO: 1.
8. The modified glucose dehydrogenase of claim 7 wherein serine 231
in the amino acid sequence shown as SEQ ID NO: 1 is replaced by
another amino acid residue.
9. The modified glucose dehydrogenase of claim 3 wherein at least
one amino acid residue is replaced by another amino acid residue in
the region defined by residues 186-221 in the amino acid sequence
shown as SEQ ID NO: 1.
10. The modified glucose dehydrogenase of claim 9 wherein an amino
acid residue corresponding to glutamine 209 in the amino acid
sequence shown as SEQ ID NO: 1 is replaced by another amino acid
residue.
11. The modified glucose dehydrogenase of claim 9 wherein an amino
acid residue corresponding to glutamate 210 in the amino acid
sequence shown as SEQ ID NO: 1 is replaced by another amino acid
residue.
12. The modified glucose dehydrogenase of claim 3 wherein at least
one amino acid residue is replaced by another amino acid residue in
the region defined by residues 412-421 in the amino acid sequence
shown as SEQ ID NO: 1.
13. The modified glucose dehydrogenase of claim 12 wherein an amino
acid residue corresponding to aspartate 420 in the amino acid
sequence shown as SEQ ID NO: 1 is replaced by another amino acid
residue.
14. The modified glucose dehydrogenase of claim 12 wherein an amino
acid residue corresponding to alanine 421 in the amino acid
sequence shown as SEQ ID NO: 1 is replaced by another amino acid
residue.
15. A glucose dehydrogenase having pyrrolo-quinoline quinone as a
coenzyme comprising the sequence: TABLE-US-00007 Asn Leu Asp Gly
Xaa23l Ile Pro Lys Asp Asn Pro Ser Phe Asn Gly Val Val Ser
wherein Xaa231 represents a natural amino acid residue other than
Ser.
16. A glucose dehydrogenase having pyrrolo-quinoline quinone as a
coenzyme comprising the sequence: TABLE-US-00008 Gly Asp Gln Gly
Arg Asn Gln Leu Ala Tyr Leu Phe Leu Pro Asn Gln Ala Gln His Thr Pro
Thr Gln Xaa209 Xaa210 Leu Asn Gly Lys Asp Tyr His Thr Tyr Met
Gly
wherein Xaa209 and Xaa210 represent any natural amino acid residue,
provided that when Xaa209 represents Gln, Xaa210 does not represent
Glu.
17. A glucose dehydrogenase having pyrrolo-quinoline quinone as a
coenzyme comprising the sequence: TABLE-US-00009 Pro Thr Tyr Ser
Thr Thr Tyr Asp Xaa420 Xaa421
wherein Xaa420 and Xaa421 represent any natural amino acid residue,
provided that when Xaa420 represents Asp, Xaa421 does not represent
Ala.
18. A gene encoding the modified glucose dehydrogenase of any one
of claim 1.
19. A vector comprising the gene of claim 18.
20. A transformant comprising the gene of claim 18.
21. The transformant of claim 20 wherein the gene is integrated
into the main chromosome.
22. A glucose assay kit comprising the modified glucose
dehydrogenase of any one of claim 1.
23. A glucose sensor comprising the modified glucose dehydrogenase
of any one of claim 1.
Description
[0001] This application is a Continuation of co-pending application
Ser. No. 09/958,231 filed on Oct. 5, 2001. Application Ser. No.
09/958,231 is the U.S. National Phase under 35 U.S.C. .sctn. 371 of
PCT International Application No. PCT/JP00/02322, filed Apr. 10,
2000, which designated the United States. This application claims
priority of Application No. 101143/1999 and 9152/2000 filed in
Japan on Apr. 8, 1999 and Jan. 18, 2000, respectively. Priority is
claimed under 35 U.S.C. .sctn. 119 and .sctn. 120 and the entire
contents of all are hereby incorporated by reference.
TECHNICAL FIELD
[0002] 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
[0003] 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.
[0004] An object of the present invention is to provide a modified
water-soluble PQQGDH with improved thermal stability.
DISCLOSURE OF THE INVENTION
[0005] We found that PQQGDHs with high stability are useful as
novel enzymes alternative to the enzymes that have been used for
enzymatic glucose assays. PQQGDHs are useful as recognition
elements of glucose sensors because they have high oxidation
activity for glucose and they are coenzyme-bound enzymes that
require no oxygen as an electron acceptor.
[0006] PQQGDHs catalyze the reaction in which glucose is oxidized
to produce gluconolactone. PQQGDHs 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. 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.
[0007] Recently, a Dutch group made an X-ray crystal structure
analysis of the water-soluble PQQGDH to show the higher-order
structure of the enzyme (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 (FIG. 7).
[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 thermal
stability, we succeeded in obtaining an enzyme with very high
stability by introducing an amino acid change into a specific
region of the water-soluble PQQGDH.
[0009] Accordingly, the present invention provides a modified
glucose dehydrogenase having pyrrolo-quinoline quinone as a
coenzyme wherein an amino acid residue corresponding to serine 231
or glutamine 209 or glutamate 210 or aspartate 420 or alanine 421
in the water-soluble PQQGDH derived from Acinetobacter
calcoaceticus (hereinafter also referred to as the wild-type
PQQGDH) is replaced by another amino acid residue. As used herein,
the "modified glucose dehydrogenase" means a glucose dehydrogenase
wherein at least one amino acid residue in a naturally occurring
glucose dehydrogenase is replaced by another amino acid residue.
The amino acid numbering herein starts from the initiator
methionine as the +1 position.
[0010] The present invention also provides a 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 one or more regions selected from the group
consisting of the regions defined by residues 48-53, 60-62, 69-71,
79-82, 91-101, 110-115, 127-135, 147-150, 161-169, 177-179,
186-221, 227-244, 250-255, 261-263, 271-275, 282-343, 349-377,
382-393, 400-403, 412-421, 427-432, 438-441 and 449-468 in the
amino acid sequence shown as SEQ ID NO: 1, characterized in that it
has higher thermal stability than that of the water-soluble PQQGDH
derived from Acinetobacter calcoaceticus. Preferably, the modified
PQQGDH of the present invention has a residual activity that is
higher than the residual activity of the wild-type PQQGDH by 10% or
more, more preferably 20% or more, still more preferably 30% or
more after heat treatment at 50.degree. C. for 10 minutes.
Preferably, the modified PQQGDH of the present invention has a heat
inactivation half-life that is longer than the heat inactivation
half-life of the wild-type PQQGDH by 5 minutes or more, more
preferably 15 minutes or more at 55.degree. C. In especially
preferred modified PQQGDHs of the present invention, at least one
amino acid residue is replaced by another amino acid residue in the
region defined by residues 227-244, 186-221 or 412-421 in the amino
acid sequence shown as SEQ ID NO: 1. In still more preferred
modified PQQGDHs of the present invention, serine 231 is replaced
by an amino acid residue selected from the group consisting of
lysine, asparagine, aspartate, histidine, methionine, leucine and
cysteine, or glutamine 209 is replaced by lysine, or glutamate 210
is replaced by lysine, or aspartate 420 is replaced by lysine, or
alanine 421 is replaced by aspartate in the amino acid sequence
shown as SEQ ID NO: 1.
[0011] In another aspect, modified PQQGDHs of the present invention
comprise the sequence: Asn Leu Asp Gly Xaa231 Ile Pro Lys Asp Asn
Pro Ser Phe Asn Gly Val Val Ser (SEQ ID NO: 3) wherein Xaa231
represents a natural amino acid residue other than Ser; or the
sequence: TABLE-US-00001 (SEQ ID NO: 4) Gly Asp Gln Gly Arg Asn Gln
Leu Ala Tyr Leu Phe Leu Pro Asn Gln Ala Gln His Thr Pro Thr Gln
Xaa209 Xaa210 Leu Asn Gly Lys Asp Tyr His Thr Tyr Met Gly
wherein Xaa209 and Xaa210 represent any natural amino acid residue,
provided that when Xaa209 represents Gln, Xaa 210 does not
represent Glu; or the sequence: Pro Thr Tyr Ser Thr Thr Tyr Asp
Xaa420 Xaa421 (SEQ ID NO: 5) wherein Xaa420 and Xaa421 represent
any natural amino acid residue, provided that when Xaa420
represents Asp, Xaa421 does not represent Ala.
[0012] 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.
[0013] Enzyme proteins of modified PQQGDHs of the present invention
have high thermal stability and high oxidation activity for glucose
so that they can be applied to highly sensitive and highly
selective glucose assays. Especially, they are expected to provide
the advantages that the enzymes can be produced at high yield with
less inactivation during preparation/purification; the enzymes can
be easily stored because of their high stability in solutions; the
enzymes can be used to prepare an assay kit or an enzyme sensor
with less inactivation; and the assay kit or enzyme sensor prepared
with the enzymes has excellent storage properties because of the
high thermal stability.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows the structure of the plasmid pGB2 used in the
present invention.
[0015] FIG. 2 shows a scheme for preparing a mutant gene encoding a
modified enzyme of the present invention.
[0016] FIG. 3 shows thermal stability of a modified enzyme of the
present invention.
[0017] FIG. 4 shows substrate specificities of modified enzymes of
the present invention.
[0018] FIG. 5 shows a glucose assay using a modified PQQGDH of the
present invention.
[0019] FIG. 6 shows a calibration curve of an enzyme sensor using a
modified PQQGDH of the present invention.
[0020] FIG. 7 shows the topology of a water-soluble GDH (Oubrie et
al., FIG. 4).
THE MOST PREFERRED EMBODIMENTS OF THE INVENTION
Structure of Modified PQQGDHs
[0021] 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 residual activity of the PQQGDHs after heat treatment to give a
number of clones that express PQQGDHs with improved thermal
stability.
[0022] Analysis of the nucleotide sequence of one of these clones
showed that Ser 231 had been changed to Cys. When this amino acid
residue was replaced by various other amino acid residues, mutant
enzymes with higher thermal stability than that of the wild type
water-soluble PQQGDH were obtained in every case.
[0023] The water-soluble PQQGDH has the structure of a
.beta.-propeller protein composed of six W-motifs. In the present
invention, it was found that thermal stability is improved by
replacing Ser 231 in the loop region defined by residues 227-244 by
another amino acid residue. Then, site-specific mutations were
introduced into other loop regions to try to improve the thermal
stability. Mutant enzymes carrying Gln209Lys or Glu210Lys in the
loop defined by residues 186-221 or Asp420Lys or Ala421Asp in the
loop defined by residues 412-421 showed improved thermal
stability.
[0024] Thus, it was demonstrated that water-soluble PQQGDHs with
improved thermal stability can be constructed by introducing a
proper change into a loop region according to the present
invention. This is probably because the interaction between the
loop regions connecting W-motifs contributes to the stabilization
of the structure of the .beta.-propeller protein in water-soluble
PQQGDHs. The residues Ser231, Gln209, Gly210, Asp420 and Ala421
shown above are only illustrative but not limiting the present
invention. The present invention first showed in the art that
thermal stability of PQQGDHs can be improved by introducing a
change into a specific site of the structural gene in a loop
region, thereby providing here a methodology for improving thermal
stability of PQQGDHs.
[0025] Modified PQQGDHs of the present invention are characterized
in that they contain an amino acid residue change in a specific
region in the amino acid sequence of the wild-type PQQGDH shown as
SEQ ID NO: 1. Accordingly, the present invention provides a
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 one or more regions selected from the
group consisting of the regions defined by residues 48-53, 60-62,
69-71, 79-82, 91-101, 110-115, 127-135, 147-150, 161-169, 177-179,
186-221, 227-244, 250-255, 261-263, 271-275, 282-343, 349-377,
382-393, 400-403, 412-421, 427-432, 438-441 and 449-468 in the
amino acid sequence shown as SEQ ID NO: 1.
[0026] In preferred modified PQQGDHs of the present invention, at
least one amino acid residue is replaced by another amino acid
residue in the region defined by residues 227-244, 186-221 or
412-421 in the amino acid sequence shown as SEQ ID NO: 1. In
especially preferred modified PQQGDHs of the present invention,
serine 231 is replaced by an amino acid residue selected from the
group consisting of lysine, asparagine, aspartate, histidine,
methionine, leucine and cysteine, or glutamine 209 is replaced by
lysine, or glutamate 210 is replaced by lysine, or aspartate 420 is
replaced by lysine, or alanine 421 is replaced by aspartate in the
amino acid sequence shown as SEQ ID NO: 1.
[0027] In another aspect, modified PQQGDHs of the present invention
comprise the sequence: TABLE-US-00002 (SEQ ID NO: 3) Asn Leu Asp
Gly Xaa231 Ile Pro Lys Asp Asn Pro Ser Phe Asn Gly Val Val Ser
[0028] wherein Xaa231 represents a natural amino acid residue other
than Ser; or the sequence: TABLE-US-00003 (SEQ ID NO: 4) Gly Asp
Gln Gly Arg Asn Gln Leu Ala Tyr Leu Phe Leu Pro Asn Gln Ala Gln His
Thr Pro Thr Gln Xaa209 Xaa210 Leu Asn Gly Lys Asp Tyr His Thr Tyr
Met Gly
wherein Xaa209 and Xaa210 represent any natural amino acid residue,
provided that when Xaa209 represents Gln, Xaa 210 does not
represent Glu; or the sequence: Pro Thr Tyr Ser Thr Thr Tyr Asp
Xaa420 Xaa421 (SEQ ID NO: 5) wherein Xaa420 and Xaa421 represent
any natural amino acid residue, provided that when Xaa420
represents Asp, Xaa 421 does not represent Ala.
[0029] 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. Various techniques for such
deletion, substitution or addition of amino acid residues 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. Those skilled in the art
can readily test whether or not a glucose dehydrogenase containing
such deletion, substitution or addition has a desired glucose
dehydrogenase activity according to the teaching herein. Those
skilled in the art can also predict a region having a loop
structure in water-soluble PQQGDHs derived from other bacteria
according to the teaching herein and replace an amino acid residue
in this region to obtain modified glucose dehydrogenases with
improved thermal stability. Particularly, an amino acid residue
corresponding to serine 231, glutamine 209, glutamate 210,
aspartate 420 or alanine 421 in the water-soluble PQQGDH derived
from Acinetobacter calcoaceticus can be readily identified by
comparing the primary structures of proteins in alignment, so that
modified glucose dehydrogenases can be obtained by replacing such a
residue by another amino acid residue according to the present
invention. These modified glucose dehydrogenases are also within
the scope of the present invention.
[0030] Process for Preparing Modified PQQGDHs
[0031] The sequence of the gene encoding the wild-type
water-soluble PQQGDH derived from Acinetobacter calcoaceticus is
defined by SEQ ID NO: 2.
[0032] Genes encoding modified PQQGDHs of the present invention can
be constructed by replacing the nucleotide sequence encoding an
amino acid residue occurring in a loop region as described above 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.
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.
[0033] Random mutations are introduced by error-prone PCR into a
target loop region to construct a gene library of modified
water-soluble PQQGDHs carrying mutations in the loop region. These
genes are transformed into E. Coli to screen each clone for the
thermal stability of the PQQGDH. 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 heated at 60-70.degree. C. for about 30
minutes and then combined with glucose and a PMS-DCIP dye to
visually determine the residual PQQGDH activity so that clones
showing residual activity even after heat treatment are selected
and analyzed for the nucleotide sequence to confirm the
mutation.
[0034] 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
[0035] PQQGDHs of the present invention associate with PQQ as a
coenzyme in catalyzing the reaction in which glucose is oxidized to
produce gluconolactone.
[0036] 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.
Thermal Stability
[0037] Thermal stability of modified PQQGDHs of the present
invention can be evaluated by incubating the enzyme of interest at
a high temperature (for example, 55.degree. C.), sampling aliquots
at regular intervals and assaying the enzyme activity to monitor
the decrease in the enzyme activity with time. Typically, thermal
stability of an enzyme is expressed as a heat inactivation
half-life, i.e. the time required for the enzyme activity to be
reduced to 50% (t.sub.1/2). Alternatively, thermal stability can
also be expressed as the residual enzyme activity after heat
treatment of the enzyme for a given period (the ratio of the
activity after heat treatment to the activity before heat
treatment).
[0038] Modified PQQGDHs of the present invention are characterized
by higher thermal stability than that of the wild-type PQQGDH.
Thus, they have the advantages that the enzymes can be produced at
high yield with less inactivation during preparation/purification;
the enzymes can be easily stored because of their high stability in
solutions; the enzymes can be used to prepare an assay kit or an
enzyme sensor with less inactivation; and the assay kit or enzyme
sensor prepared with the enzymes has excellent storage properties
because of the high thermal stability.
Glucose Assay Kit
[0039] 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
[0040] 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.
[0041] 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.
[0042] 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. 1999-101143 and 2000-9152, the disclosure of which is entirely
incorporated herein as reference.
[0043] 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
[0044] 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 the coding region 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-00004 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 1M
.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
[0045] The resulting mutant water-soluble PQQGDH library was
transformed into E. coli and each colony formed was transferred to
a microtiter plate. After heating the plate at 60.degree. C. for
about 30 minutes, glucose and PMS-DCIP were added and the residual
PQQGDH activity was visually evaluated. A number of clones showing
PQQGDH activity even after heat treatment were obtained.
[0046] One of these clones was randomly selected and analyzed for
the nucleotide sequence to show that serine 231 had been changed to
cysteine.
EXAMPLE 2
Construction of Modified PQQGDH Genes
[0047] Based on the structural gene of the PQQGDH derived from
Acinetobacter calcoaceticus shown as SEQ ID NO: 2, the nucleotide
sequence encoding serine 231, glutamine 209, aspartate 420 or
alanine 421 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, "S231D" means that serine 231 is
replaced by aspartate, for example. TABLE-US-00005 TABLE 2 (SEQ ID
NOS: 6-16) S231D 5'-C CTT TGG AAT ATC TCC ATC AAG ATT TAA GC-3'
S231H 5'-C CTT TGG AAT ATG TCC ATC AAG ATT TAA GC-3' S231K 5'-C CTT
TGG AAT TTT TCC ATC AAG ATT TAA GC-3' S231L 5'-C CTT TGG AAT CAT
TCC ATC AAG ATT TAA GC-3' S231M 5'-C CTT TGG AAT AGT TCC ATC AAG
ATT TAA GC-3' S231N 5'-C CTT TGG AAT ATT TCC ATC AAG ATT TAA GC-3'
I278F 5'-C AAT GAG GTT GAA TTC ATC GTC AGA G-3' Q209K 5'-C ACC ATT
CAG TTC TTT TTG AGT TGG C-3' E210K 5'-C ACC ATT CAG TTT TTG TTG AGT
TGG C-3' D420K 5'-A CAT CGG TAC AGC TTT ATC ATA AGT AG-3' A421D
5'-A CAT CGG TAC ATC GTC ATC ATA AGT AG-3'
[0048] 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 .mu.l of T4
DNA polymerase and 5 .mu.l of sterilized water to synthesize a
complementary strand.
[0049] The synthetic strand was transformed into a DNA mismatch
repair-deficient strain E. coli BMH71-18mutS and shake-cultured
overnight to amplify the plasmid.
[0050] 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.
EXAMPLE 3
Preparation of Modified Enzymes
[0051] 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.
[0052] 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 recovered
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 4
Assay of Enzyme Activity
[0053] 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 5
Evaluation of Thermal Stability of Crude Enzyme Samples
[0054] Each of the crude enzyme samples of the wild-type and
modified PQQGDHs obtained in Example 3 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 then incubated at 55.degree. C. Aliquots were
sampled at regular intervals and rapidly cooled on ice. These
samples were assayed for the enzyme activity by the method of
Example 4 to determine the time required for reducing the activity
to 50% (t.sub.1/2).
[0055] The results are shown in Table 3. TABLE-US-00006 TABLE 3
t.sub.1/2 (min) Wild type 10 S231K 95 S231L 16 S231D 25 S231C 50
S231M 14 S231H 15 S231N 50 I278F 25 Q209K 40 E210K 40 D420K 20
A421D 80
[0056] All the modified PQQGDHs of the present invention have a
heat inactivation half-life at 55.degree. C. longer than that of
the wild-type PQQGDH, showing that they have higher thermal
stability than that of the wild-type PQQGDH.
EXAMPLE 6
Evaluation of Thermal Stability of Purified Enzyme Samples
[0057] The purified samples of the wild-type enzyme and the
modified enzyme S231K obtained in Example 3 were measured for the
heat inactivation half-life at 55.degree. C. in the same manner as
in Example 5. The purified samples of the wild-type enzyme and the
modified enzyme S231K had half-lives of 5 minutes and 41 minutes,
respectively.
[0058] Then, each of the purified samples of the wild-type enzyme
and the modified enzyme S231K obtained in Example 3 was converted
into a holoenzyme in the presence of 1 .mu.M PQQ and 1 mM
CaCl.sub.2 for 1 hour or longer. Then, each sample was incubated at
a given temperature in 10 mM MOPS buffer (pH 7.0) containing 1
.mu.M PQQ and 1 mM CaCl.sub.2 for 10 minutes, and then rapidly
cooled on ice. These samples were assayed for the enzyme activity
by the method of Example 4 to determine the residual activity
relative to the activity before heat treatment.
[0059] The results are shown in FIG. 3. The modified enzyme S231K
had higher activities than those of the wild-type enzyme at various
temperatures of 40-62.5.degree. C.
EXAMPLE 7
Evaluation of Enzyme Activity
[0060] The crude enzyme sample of the modified enzyme S231K
obtained in Example 3 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 .mu.l of 10 mM phosphate buffer, pH 7.0) and 10 .mu.l of
glucose solutions at various concentrations, and assayed for the
enzyme activity at room temperature by the method shown in Example
4. The Km and Vmax were determined by plotting the substrate
concentration vs. enzyme activity. The S231K variant had a Km value
for glucose of about 20 mM and a Vmax value of 3300 U/mg. The Km
value of the wild-type PQQGDH for glucose reported to date was
about 20 mM with the Vmax value being 2500-7000 U/mg depending on
the measurement conditions. These results show that the modified
PQQGDH S231K has high activity comparable to that of the wild-type
PQQGDH.
EXAMPLE 8
Evaluation of Substrate Specificity
[0061] Crude samples of various modified enzymes were tested for
substrate specificity. The substrates tested were glucose,
2-deoxy-D-glucose, mannose, allose, 3-o-methyl-D-glucose,
galactose, xylose, lactose and maltose, and each sample was
incubated with 20 mM of each substrate for 30 minutes in the
presence of 1 .mu.M PQQ and 1 mM CaCl.sub.2 and assayed for the
enzyme activity in the same manner as in Example 7 to determine the
relative activity to the activity for glucose. As shown in FIG. 4,
all the modified enzymes of the present invention showed a similar
substrate specificity to that of the wild-type enzyme.
EXAMPLE 9
Glucose Assay
[0062] A modified PQQGDH was used for assaying glucose. The
modified enzyme S231K 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 4 based on changes of the
absorbance of DCIP at 600 nm. As shown in FIG. 5, the modified
PQQGDH S231K can be used for assaying glucose in the range of 5
mM-50 mM.
EXAMPLE 10
Preparation and Evaluation of an Enzyme Sensor
[0063] Five units of the modified enzyme S231K was 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.
[0064] Thus prepared enzyme sensor was used to measure glucose
levels. FIG. 6 shows the resulting calibration curve. Thus, the
enzyme sensor having a modified PQQGDH of the present invention
immobilized thereon could be used for assaying glucose in the range
of 1 mM-12 mM.
INDUSTRIAL APPLICABILITY
[0065] Modified PQQGDHs of the present invention have excellent
thermal stability so that they are expected to provide the
advantages that the enzymes can be produced at high yield with less
inactivation during preparation/purification; the enzymes can be
easily stored because of their high stability in solutions; the
enzymes can be used to prepare an assay kit or an enzyme sensor
with less inactivation; and the assay kit or enzyme sensor prepared
with the enzymes has excellent storage properties because of the
high thermal stability.
Sequence CWU 1
1
16 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 18 PRT Acinetobacter
calcoaceticus UNSURE 5 Xaa is any amino acid residue 3 Asn Leu Asp
Gly Xaa Ile Pro Lys Asp Asn Pro Ser Phe Asn Gly Val 1 5 10 15 Val
Ser 4 36 PRT Acinetobacter calcoaceticus UNSURE 24 Xaa is any amino
acid residue 4 Gly Asp Gln Gly Arg Asn Gln Leu Ala Tyr Leu Phe Leu
Pro Asn Gln 1 5 10 15 Ala Gln His Thr Pro Thr Gln Xaa Xaa Leu Asn
Gly Lys Asp Tyr His 20 25 30 Thr Tyr Met Gly 35 5 10 PRT
Acinetobacter calcoaceticus UNSURE 9 Xaa is any amino acid residue
5 Pro Thr Tyr Ser Thr Thr Tyr Asp Xaa Xaa 1 5 10 6 30 DNA
Artificial Sequence primer for point mutation 6 cctttggaat
atctccatca agatttaagc 30 7 30 DNA Artificial Sequence primer for
point mutation 7 cctttggaat atgtccatca agatttaagc 30 8 30 DNA
Artificial Sequence primer for point mutation 8 cctttggaat
ttttccatca agatttaagc 30 9 30 DNA Artificial Sequence primer for
point mutation 9 cctttggaat cattccatca agatttaagc 30 10 30 DNA
Artificial Sequence primer for point mutation 10 cctttggaat
agttccatca agatttaagc 30 11 30 DNA Artificial Sequence primer for
point mutation 11 cctttggaat atttccatca agatttaagc 30 12 26 DNA
Artificial Sequence primer for point mutation 12 caatgaggtt
gaattcatcg tcagag 26 13 26 DNA Artificial Sequence primer for point
mutation 13 gaccattcag ttctttttga gttggc 26 14 26 DNA Artificial
Sequence primer for point mutation 14 gaccattcag tttttgttga gttggc
26 15 27 DNA Artificial Sequence primer for point mutation 15
acatcggtac agctttatca taagtag 27 16 27 DNA Artificial Sequence
primer for point mutation 16 acatcggtac atcgtcatca taagtag 27
* * * * *