JPH11243949A - Glucose dehydrogenase having pqq as prosthetic group and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new glucose dehydrogenase producible in a mass at a low cost by gene recombination technique and useful for the determination of blood sugar level, etc. SOLUTION: This enzyme is (A) a protein composed of the amino acid sequence described by formula or (B) a protein composed of the amino acid sequence of formula provided that one or several amino acid sequences are deleted, substituted or added and having glucose dehydrogenase activity. The glucose dehydrogenase having PQQ as prosthetic group is produced by culturing a PQQ-producing transformed microorganism obtained by transforming a microorganism such as Pseudomonas aeruginosa with a recombinant vector integrated with a DNA fragment containing a gene coding for the enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel glucose dehydrogenase (hereinafter referred to as G) having PQQ as a prosthetic group.
DH), a gene encoding the GDH, the GD
H. A recombinant vector comprising a gene fragment encoding H, a transformant obtained by transforming a microorganism having PQQ-producing ability with the recombinant vector, a method for producing GDH by culturing the transformant, and a method for producing GDH. The present invention relates to a stabilizing method and a GDH composition stabilized by the stabilizing method.

[0002]

2. Description of the Related Art GDH was established in 1959 by J. Norway.
Discovered by G. Hauge as an enzyme with an unknown quinone compound. On the other hand, PQQ (Pyrrolo Quinoline Quin
one) is the third coenzyme of dehydrogenase, whose chemical structure was determined in 1979. It uses methanol dehydrogenase of methanol assimilating bacteria, alcohol dehydrogenase of acetic acid bacteria and glucose dehydrogenase. Mainly, in many organisms, its presence is confirmed mainly by dehydrogenase.

[0003] Since these dehydrogenases can reduce artificial electron acceptors, they can be detected with visible light and with high sensitivity by using a dye such as nitroblue tetrazolium, and the equilibrium reaction such as NAD-dependent dehydrogenase can be performed. It is considered to be extremely useful for the quantification of trace amounts of compounds because it is a one-way reaction instead of (Minami Ameyama, Methods in
Enzymol. 89, 20 (1982)).

Among the enzymes having PQQ as a prosthetic group, the most useful is PQQ-dependent GDH, which can be used for measuring blood glucose. As for actual use, it can be used widely as a normal biochemical reagent, as well as a color reaction of a dry reagent immobilized on a membrane or a sensor immobilized on a chip. Compared to glucose oxidase and NAD (P) -dependent GDH which act on glucose in the same way, it is not affected by dissolved oxygen,
The feature is that the device can be made simple and inexpensive because the reaction is simple.

The cloning of GDH having PQQ as a prosthetic group was performed by Acinetobacter calcoaceticus LMD79.41 (A.-M.Cle
ton-Jansen et al., J. Bacteriol., 170, 2121 (19
88) and Mol. Gen. Genet., 217, 430 (198).
9)), Escherichiacoli (A.
-M. Cleton-Jansen et al., J. Bacteriol., 172, 6308
(1990)), Gluconobacter oxydans (Gl
uconobacter oxydans) (Mol. Gen. Genet., 229, 2)
06 (1991)), and expression in E. coli has also been confirmed.

[0006]

It is known that enterobacteria such as Escherichia coli, which has been genetically well analyzed and optimized as a transforming host, do not produce PQQ. GD with PQQ as the prosthetic group
It is known that Escherichia coli is transformed with a vector into which a gene fragment encoding H has been incorporated, and only the apo-type GDH having no activity can be obtained by culturing the Escherichia coli. These apo-type GDHs can be converted into active holo-type GDH by adding PQQ from outside, but the required PQQ is very expensive as a reagent. Further, it has been confirmed that not all apo-forms are converted to holo-forms on an industrial scale.

Also, Biotechnol. Lett., 16, 12, 1
265 (1994), when culturing transformed E. coli,
It has been reported that PQQ is added to a medium to produce active holo-type GDH, but also in this case, a large amount of expensive PQQ must be used. Furthermore, Mol. Gen. Genet., 2
29, 206 (1991), a gene fragment encoding GDH of Gluconobacter oxydans is expressed by integrating it into the chromosome of Gluconobacter oxydans itself. In this case, the gene fragment is present on the chromosome, The expression level is small because it is not a multiple copy.

[0008]

Means for Solving the Problems As a result of various studies to achieve the above object, the present inventors have found that a recombinant vector comprising a DNA fragment containing a gene encoding GDH having PQQ as a prosthetic group is incorporated. By culturing a transformed microorganism obtained by transforming a microorganism having PQQ-producing ability by using the method described above, and collecting GDH having PQQ as a prosthetic group from the culture, whereby GDH can be mass-produced at low cost. Reached the present invention.

That is, the present invention provides that the present invention is obtained by transforming a microorganism capable of producing PQQ with a recombinant vector into which a DNA fragment containing a gene encoding GDH having PQQ as a prosthetic group is incorporated. A method for producing GDH, comprising culturing a transformed microorganism, and collecting GDH having PQQ as a prosthetic group from the culture.

[0010] The present invention is a glucose dehydrogenase having PQQ, a protein of the following (a) or (b), as a prosthetic group: (A) a protein consisting of the amino acid sequence described in Sequence Listing / SEQ ID NO: 1 (b) an amino acid sequence (a) comprising one or several amino acid sequences deleted, substituted or added, and TECHNICAL FIELD The present invention relates to a glucose dehydrogenase having PQQ, which is a protein having the amino acid sequence described in Sequence Listing and SEQ ID NO: 1, as a prosthetic group.

The present invention is a glucose dehydrogenase having PQQ, which is a protein of the following (e) or (f), as a prosthetic group. (E) a protein comprising the amino acid sequence described in SEQ ID NO: 3 (f) an amino acid sequence (e) wherein one or several amino acid sequences are deleted, substituted or added, and TECHNICAL FIELD The present invention relates to a glucose dehydrogenase having PQQ, which is a protein having the amino acid sequence shown in SEQ ID NO: 3 in the Sequence Listing and having a prosthetic group, as a prosthetic group.

The present invention is a gene encoding a glucose dehydrogenase having PQQ, a protein of the following (a) or (b), as a prosthetic group: (A) a protein consisting of the amino acid sequence described in Sequence Listing / SEQ ID NO: 1 (b) an amino acid sequence (a) comprising one or several amino acid sequences deleted, substituted or added, and TECHNICAL FIELD The present invention is a gene encoding a glucose dehydrogenase having PQQ, which is a protein consisting of the amino acid sequence shown in Sequence Listing and SEQ ID NO: 1, as a prosthetic group.

The present invention relates to a gene encoding glucose dehydrogenase having PQQ, which is a protein of the following (c) or (d), as a prosthetic group. (C) DNA consisting of the nucleotide sequence described in SEQ ID NO: 2 (d) In the above sequence (c), one or several bases are deleted, substituted or added, and glucose dehydrogenase activity TECHNICAL FIELD The present invention is a gene encoding glucose dehydrogenase having PQQ having a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 as a prosthetic group.

The present invention is a gene encoding a glucose dehydrogenase having PQQ, a protein of the following (e) or (f), as a prosthetic group. (E) a protein comprising the amino acid sequence described in SEQ ID NO: 3 (f) an amino acid sequence (e) wherein one or several amino acid sequences are deleted, substituted or added, and TECHNICAL FIELD The present invention is a gene encoding a glucose dehydrogenase having PQQ, which is a protein consisting of the amino acid sequence shown in Sequence Listing and SEQ ID NO: 3, as a prosthetic group.

The present invention relates to a gene encoding glucose dehydrogenase having PQQ, a protein of the following (g) or (h), as a prosthetic group. (G) DNA consisting of the base sequence described in SEQ ID NO: 4 (h) In the above sequence (g), one or several bases are deleted, substituted or added, and glucose dehydrogenase activity TECHNICAL FIELD The present invention is a gene encoding glucose dehydrogenase having PQQ having a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 4 as a prosthetic group.

The present invention is a recombinant vector containing a gene encoding glucose dehydrogenase having PQQ as a prosthetic group. The present invention is the above-mentioned recombinant vector, wherein a DNA fragment containing a gene encoding glucose dehydrogenase having PQQ as a prosthetic group is integrated and can be replicated in a microorganism capable of producing PQQ.

[0017] The present invention provides a recombinant vector comprising PQQ
It is a transformant obtained by transforming a microorganism having productivity. The present invention is the above transformant, wherein the glucose dehydrogenase having PQQ as a prosthetic group is derived from Acinetobacter calcoaceticus or Acinetobacter baumannii. The present invention is the above transformant, wherein the microorganism having PQQ-producing ability is a microorganism belonging to the genus Pseudomonas or the genus Acinetobacter.

According to the present invention, the microorganism having the ability to produce PQQ is Pseudomonas aeruginos
a), Pseudomonas fluorescens
fluorescens), Pseudomon
as putida), Acinetobacter calcoaceticus, and Acinetobacter baumannii. The above transformant which is a microorganism selected from the group consisting of Acinetobacter baumannii. The present invention relates to a method for producing PQQ-producing microorganisms comprising Pseudomonas sp.
The above transformant which is Pseudomonas putida. The present invention relates to a method wherein the microorganism having PQQ-producing ability is Acinetobacter calcoaceticas.
oaceticus) or Acinetobacter baumannii. The present invention is the above transformant, in which glucose dehydrogenase having PQQ as a prosthetic group is soluble.

The present invention relates to a DN containing a gene encoding glucose dehydrogenase having PQQ as a prosthetic group.
Production of glucose dehydrogenase by culturing a transformed microorganism in which a microorganism having PQQ-producing ability has been transformed with a recombinant vector incorporating the A fragment, and collecting glucose dehydrogenase having PQQ as a prosthetic group from the culture Is the way.

According to the present invention, glucose dehydrogenase having PQQ as a prosthetic group is Acinetobacter calcoaceticus or Acinetobacter baumani.
nii) A method for producing the above-mentioned glucose dehydrogenase, which is a microorganism derived from the present invention. The present invention is the method for producing glucose dehydrogenase, wherein the microorganism having PQQ-producing ability is a microorganism belonging to the genus Pseudomonas or the genus Acinetobacter.

According to the present invention, the microorganism having the ability to produce PQQ is Pseudomonas aeruginos
a), Pseudomonas fluorescens
fluorescens), Pseudomon
asputida), Acinetobacter calcoaceticus, and Acinetobacter baumannii, a method for producing the above glucose dehydrogenase, which is a microorganism selected from the group consisting of Acinetobacter baumannii. The present invention relates to a method for producing a PQQ-producing microorganism comprising Pseudomonas puti.
da) is a method for producing the above glucose dehydrogenase. The present invention relates to a method for producing PQQ-producing microorganisms using Acinetobacter cacoaceticus.
lcoaceticus) or Acinetobacter baumannii. The present invention is the method for producing glucose dehydrogenase, wherein glucose dehydrogenase having PQQ as a prosthetic group is soluble.

The present invention provides a method for stabilizing glucose dehydrogenase, comprising freeze-drying glucose dehydrogenase having PQQ as a prosthetic group in the presence of a GOOD buffer. The present invention is a method for stabilizing the above glucose dehydrogenase in which calcium coexists. In the present invention, the GOOD buffer is PIPE
This is a method for stabilizing glucose dehydrogenase, which is a buffer selected from the group consisting of S, MES, and MOPS.

The present invention provides a stabilized glucose dehydrogenase composition characterized in that glucose dehydrogenase having PQQ as a prosthetic group is freeze-dried and maintained in the presence of a GOOD buffer. .
The present invention is the above glucose dehydrogenase composition in which calcium coexists. The present invention is the above glucose dehydrogenase composition, wherein the GOOD buffer is a buffer selected from the group consisting of PIPES, MES, and MOPS.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION A DNA fragment containing a gene encoding GDH having PQQ as a prosthetic group according to the present invention is GD
It can be obtained from H-producing bacteria. As the GDH-producing bacteria, specifically, for example, Acinetobacter calcoaceticus, Acinetobacter baumannii (Acinetobacca)
ter baumannii), Pseudomonas aeruginosa (Pseu)
domonasaeruginosa), Pseud (Pseud)
omonas putida), oxidizing bacteria such as Pseudomonas fluorescens, and Gluconobacter oxydans, Agrobacterium radiobacter, Escherichia coli, and Klebsiella aerojeans.
aerogenes). Among them, soluble GDH of Acinetobacter calcoaceticus or Acinetobacter baumannii is preferable.

The GDH-encoding gene may be extracted from these strains or may be chemically synthesized. Furthermore, by utilizing the PCR method, DQ containing a glucose dehydrogenase gene having PQQ as a prosthetic group is obtained.
It is also possible to obtain NA fragments.

The genes encoding GDH include:
For example, (a) a gene encoding a protein consisting of the amino acid sequence described in SEQ ID NO: 1 or (b) an amino acid sequence (a) in which one or several amino acid sequences are deleted, substituted or added. And a gene encoding glucose dehydrogenase having PQQ, which is a protein having the above amino acid sequence and having glucose dehydrogenase activity, as a prosthetic group.

Further, (e) a gene encoding a protein consisting of the amino acid sequence described in SEQ ID NO: 3 or (f) one or several amino acid sequences in the amino acid sequence (e) are deleted, A gene consisting of a substituted or added amino acid sequence and encoding glucose dehydrogenase having PQQ, a protein having glucose dehydrogenase activity, as a prosthetic group can also be mentioned.

Further, (c) a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 or (d) one or several bases deleted in the above sequence (c);
DN which is substituted or added and encodes a protein having glucose dehydrogenase activity
There is A.

Further, (g) a DNA consisting of the base sequence described in SEQ ID NO: 4 or (h) one or several bases deleted in the above sequence (g);
DN which is substituted or added and encodes a protein having glucose dehydrogenase activity
There is A.

In the present invention, a method for obtaining a gene encoding GDH includes the following method.
For example, Acinetobacter calcoaceticus NCIB1151
After isolating and purifying the chromosome 7, the DNA was cut using sonication, restriction enzyme treatment, etc., and the linear expression vector was ligated and closed with DNA ligase at the blunt or cohesive ends of both DNAs. Construct a recombinant vector. After transferring the recombinant vector into a replicable host microorganism, screening is performed using the vector marker and the expression of enzyme activity as indices, and the recombinant vector containing the gene encoding GDH with PQQ as the prosthetic group is retained. Get the microorganisms that work.

Next, the microorganism carrying the recombinant vector is cultured, the recombinant vector is separated and purified from the cells of the cultured microorganism, and the gene encoding GDH can be collected from the expression vector. . For example, the gene donor Acinetobacter calcoaceticus NC
The chromosomal DNA of IB11517 is specifically collected as follows.

A culture solution obtained by stirring and culturing the gene-donating microorganism for 1 to 3 days, for example, is collected by centrifugation, and then lysed to thereby lyse the lysate. Can be prepared. As a lysis method, for example, treatment is performed with a lytic enzyme such as lysozyme, and a protease, another enzyme, or a surfactant such as sodium lauryl sulfate (SDS) is used as needed. Further, the method may be combined with a physical crushing method such as freeze-thawing or French pressing.

From the lysate obtained as described above, DN
A can be separated and purified according to a conventional method, for example, by appropriately combining methods such as protein removal treatment by phenol treatment or protease treatment, ribonuclease treatment, and alcohol precipitation treatment.

The method of cutting DNA isolated and purified from microorganisms can be performed by, for example, ultrasonic treatment, restriction enzyme treatment, or the like. Preferably, a type II restriction enzyme that acts on a specific nucleotide sequence is suitable.

As a vector for cloning,
Those constructed for gene recombination from phages or plasmids capable of autonomous growth in a host microorganism are suitable. As the phage, for example, when Escherichia coli is used as a host microorganism, Lambda gt10, Lambda gt1
1 and the like. As a plasmid, for example, when Escherichia coli is used as a host microorganism,
Examples are pBR322, pUC19, pBluescript and the like.

At the time of cloning, the vector as described above can be obtained by cutting the above-mentioned vector with the restriction enzyme used for cutting the above-mentioned GDH-encoding gene donor microbial DNA. DNA
It is not necessary to use the same restriction enzyme as the restriction enzyme used for the cleavage of DNA. The method for binding the microorganism DNA fragment to the vector DNA fragment may be any method using a known DNA ligase. For example, after annealing the cohesive end of the microbial DNA fragment and the cohesive end of the vector fragment, appropriate DIG
A recombinant vector of a microorganism DNA fragment and a vector DNA fragment is prepared by using NA ligase. If necessary, after annealing, it can be transferred to a host microorganism and a recombinant vector can be prepared using in vivo DNA ligase.

The host microorganism used for cloning is not particularly limited as long as the recombinant vector is stable, can be autonomously propagated, and can express a foreign gene. Generally, Escherichia coli W3110, Escherichia coli C600, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli DH5α and the like can be used.

As a method for transferring the recombinant vector to the host microorganism, for example, when the host microorganism is Escherichia coli, a competent cell method or an electroporation method by calcium treatment can be used.

The microorganism, which is a transformant obtained as described above, is cultured in a nutrient medium to produce a large amount of GD.
H can be produced stably. Selection of the presence or absence of the transfer of the target recombinant vector into the host microorganism depends on the target DNA.
Microorganisms that simultaneously express GDH activity by the addition of a drug resistance marker and a PQQ of a vector that retains the DNA may be searched. For example, a microorganism which grows on a selective medium based on a drug resistance marker and produces GDH may be selected.

The nucleotide sequence of the GDH gene obtained by the above method and having PQQ as a prosthetic group is described in Science, Second Edition.
14, 1205 (1981). The amino acid sequence of GDH was deduced from the nucleotide sequence determined as described above.

As described above, the PQQ once selected
Transfer from a recombinant vector carrying a GDH gene having a prosthetic group to a recombinant vector capable of replicating in a PQQ-producing microorganism can be carried out by a restriction enzyme or PCR method from a recombinant vector carrying the GDH gene. It can be easily carried out by recovering DNA as a gene and ligating it to another vector fragment. The transformation of a microorganism having PQQ-producing ability with these vectors can be performed by a competent cell method or an electroporation method using calcium treatment.

Microorganisms having the ability to produce PQQ include methanol assimilating bacteria such as Methylobacterium, acetic acid bacteria belonging to the genus Acetobacter and Gluconobacter, and flavobacterium ( Flavobacterium), Pseudomonas, Acinetobacter and the like. Among them, a host-vector system in which Pseudomonas bacteria and Acinetobacter bacteria can be used has been established and is easy to use.

Pseudomonas bacteria include Pseudomonas aeruginosa, Pseudomonas fluorescens,
Pseudomonas putida and the like can be used. In addition, Acinetobacter calcoaceticus, Acinetobacter baumannii and the like can be used as Acinetobacter bacteria.

As a recombinant vector capable of replicating in the above microorganism, a vector derived from RSF1010 or a vector having a replicon similar thereto can be used for Pseudomonas bacteria. For example, pKT240, pMMB24, etc. (MMBa
gdasarian et al., Gene, 26, 273 (1983)), pC
N40, pCN60, etc. (CCNieto et al., Gene, 87, 145)
(1990)) and pTS1137. Also, pME290 and the like (Y. Itoh et al., Gene, 36, 27 (198)
5)), pNI111, pNI20C (N. Itoh et al., J. Biochem., 11)
0,614 (1991)).

For bacteria belonging to the genus Acinetobacter, pWM43 and the like (W. Minas et al., Appl. Environ. Microbiol., 59, 28)
07 (1993)), pKT230, pWH1266, etc. (M. Hunger
Gene, 87, 45 (1990)) is available as a vector.

The culture form of the host microorganism, which is a transformant, may be selected in consideration of the nutritional and physiological properties of the host, and in most cases, liquid culture is used. Industrially, it is advantageous to carry out aeration stirring culture.

As the nutrient of the medium, those commonly used for culturing microorganisms can be widely used. The carbon source may be any carbon compound that can be assimilated, and for example, glucose, sucrose, lactose, maltose, lactose, molasses, pyruvic acid and the like are used. The nitrogen source may be any nitrogen compound that can be used, and examples thereof include peptone, meat extract, yeast extract, casein hydrolyzate, and soybean meal alkaline extract. In addition, salts such as phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary.

The culture temperature can be appropriately changed within the range in which the bacteria grow and produce GDH. In the case of the microorganism having the PQQ-producing ability as described above, the temperature is preferably about 20 to 42 ° C. The cultivation time varies somewhat depending on the conditions, but the cultivation may be completed at an appropriate time in consideration of the time when GDH reaches the maximum yield, and is usually about 12 to 72 hours. The pH of the medium can be appropriately changed within a range in which the bacteria grow and produce GDH, but is preferably in a range of about pH 6.0 to 9.0.

The culture solution containing the cells producing GDH in the culture can be directly collected and used. However, in general, when GDH is present in the culture solution, filtration and centrifugation are performed according to a conventional method. It is used after separating the GDH-containing solution and the microbial cells by, for example, When GDH is present in the cells, the cells are collected from the obtained culture by means such as filtration or centrifugation, and then the cells are disrupted by a mechanical method or an enzymatic method such as lysozyme. If necessary, a chelating agent such as EDTA and a surfactant are added to solubilize GDH, and the GDH is separated and collected as an aqueous solution.

The GDH-containing solution obtained as described above is concentrated under reduced pressure, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate or the like, or fractional precipitation with a hydrophilic organic solvent such as methanol, ethanol, acetone or the like. What is necessary is just to precipitate by the method. Heat treatment and isoelectric point treatment are also effective purification means. afterwards,
Purified GDH can be obtained by performing gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, or affinity chromatography.

For example, gel filtration using Sephadex gel (Pharmacia Biotech), DE
AE Sepharose CL-6B (Pharmacia Biotech),
Separation and purification by column chromatography such as octyl sepharose CL-6B (Pharmacia Biotech) can yield a purified enzyme preparation. The purified enzyme preparation is preferably purified to such an extent that it shows a single band by electrophoresis (SDS-PAGE).

The purified enzyme obtained as described above can be distributed as a powder by freeze drying, vacuum drying, spray drying, or the like. At this time, the purified enzyme that is dissolved in a phosphate buffer, a Tris-HCl buffer, or a GOOD buffer can be used. Preferred are GOOD buffers, especially PIPES,
MES or MOPS buffers are particularly preferred. Also,
Calcium ions or salts thereof, and glutamic acid,
GDH can be further stabilized by adding amino acids such as glutamine and lysine and further serum albumin and the like.

An example of the GDH of the present invention using PQQ as a prosthetic molecule has the following physicochemical properties. Action: D-glucose + artificial electron acceptor → δ-
Gluconolactone + reduced electron acceptor Thermal stability: about 50 ° C. or less (treatment at pH 7.5 for 30 minutes) pH stability: 3.5 to 8.5 (treatment at 25 ° C. for 16 hours) Optimum temperature: about 40 ° C Optimum pH: 7.0 Molecular weight: 50,000

In the present invention, GDH which is a prosthetic group
The activity is measured under the following conditions.

<Reagent> 50 mM PIPES buffer (pH 6.5) 0.2 mM PMS 0.2 mM NTB 30.6 mM Glucose 0.19% Triton X-100

<Measurement conditions> 3 ml of the above reagent mixture was mixed at 37 ° C.
After preliminarily heating for about 5 minutes, 0.1 ml of the enzyme solution was added and mixed gently. After that, recording was performed for 5 minutes with a spectrophotometer controlled at 37 ° C. using water as a control. Measure the change in absorbance. In the blind test, distilled water is added to the reagent mixture instead of the enzyme solution, and the absorbance change is measured in the same manner. The amount of the enzyme that produces 1/2 μmol of diformazan per minute under the above conditions is defined as 1 unit (U).

[0057]

The present invention will be described below in detail with reference to examples. Example 1 Isolation of Chromosomal DNA The chromosomal DNA of Acinetobacter calcoaceticus NCIB11517 was isolated by the following method. 100 ml of the same strain
After shaking culture in an LB medium at 30 ° C. overnight, the cells were collected by centrifugation (8000 rpm, 10 minutes). 20%
Sucrose, 50 mM Tris / HCl buffer (pH
7.6) Suspended in 5 ml of a solution containing 1 mM EDTA, 1 ml of proteinase K solution (100 mg / m
l) and keep the mixture at 37 ° C for 30 minutes.
Of 10% sodium lauroyl sarcosine solution was added.

An equal volume of a chloroform / phenol solution (1: 1) was added to the above solution, and mixed by stirring.
m, and the mixture was separated into an aqueous layer and a solvent layer by centrifugation for 3 minutes, and the aqueous layer was separated. Twice the amount of ethanol was gently overlaid on the aqueous layer, and the DNA was wound around a glass rod while slowly stirring with a glass rod to separate the DNA. This was mixed with a Tris / HCl buffer containing 1 mM EDTA (pH 8.0; hereinafter abbreviated as TE).
And dissolved. After treating this with an equal volume of a chloroform / phenol solution, the aqueous layer is separated by centrifugation, twice the amount of ethanol is added, and the DNA is separated again by the above method.
Dissolved in ml of TE.

Example 2 Preparation of DNA Fragment Containing a Gene Encoding Soluble GDH Having PQQ as a Prosthetic Group and Recombinant Vector Having the DNA Fragment 5 μg of the DNA obtained in Example 1 was digested with the restriction enzyme Sau3AI (Toyobo Co., Ltd.) After partial digestion into fragments of 2 kbp or more,
pBluescript KS (+) 1μ cut with mHI (Toyobo)
g and 1 unit of T4 DNA ligase (Toyobo) 16
The reaction was carried out at 16 ° C. for 16 hours to ligate the DNA. Connected D
NA was transformed using competent cells of Escherichia JM109 (manufactured by Toyobo). Approximately 10 ⁵ transformant colonies were obtained per μg of DNA used.

Next, the obtained colony was 50 μg / m
The cells were cultured in an LB medium containing 1 ampicillin at 30 ° C. for 24 hours, lysozyme was crushed, PQQ was added, and the soluble GDH activity of the crude enzyme solution was measured. As a result, one stock PQ
A GDH-producing strain having Q as a prosthetic group was found. The plasmid contained in the strain had an inserted DNA of about 8 kbp, and this plasmid was designated as pPGH1.

The plasmid DNA (5 μg) was digested with the restriction enzyme MboI.
The fragment was cut with I (Toyobo) and subjected to agarose gel electrophoresis to cut out a fragment of about 1.9 kb in length containing the soluble GDH gene. The isolated DNA and 1 μg of pBluescriptKS (+) cut with EcoRV (manufactured by Toyobo) were mixed with T4D.
The reaction was carried out with 1 unit of NA ligase at 16 ° C. for 16 hours.
A was connected. The ligated DNA is Escherichia coli JM
Transformation was performed using 109 competent cells. A colony having a plasmid having the GDH gene was found in the obtained colonies, and the plasmid was named pPGH2.

Example 3 Determination of Nucleotide Sequence A deletion mutant was prepared from the inserted DNA fragment of pPGH2 according to a conventional method. Various subclones can be prepared in a conventional manner using a sequencing kit (Radioactive Sequencing).
base kit) (Toyobo Co., Ltd.). The determined nucleotide sequence and amino acid sequence are as shown in SEQ ID NO: 1.
And 2. The molecular weight of the protein determined from the amino acid sequence was about 52800, which was almost the same as the molecular weight of GDH of Acinetobacter calcoaceticus NCIB11517.

Example 4: Acinetobacter baumannii
Determination of base sequence of soluble GDH of JCM6841 Chromosomal DNA of Acinetobacter baumannii JCM6841
Was isolated in the same manner as in Example 1, and DN containing a gene encoding soluble GDH having PQQ as a prosthetic group.
Preparation of the recombinant vector having the A fragment and the DNA fragment was carried out in the same manner as in Example 2 to obtain pPGH6 having an insert fragment of about 7 Kbp. 4 kb DN containing a DNA fragment containing the gene encoding GDH from the plasmid
The fragment A was subcloned by a conventional method to construct pPGH7.

Next, a deletion mutant was prepared for the inserted DNA fragment of pPGH7 according to a conventional method. The nucleotide sequences of the various subclones were determined using a sequencing kit (Radioactive Sequencing Kit) (manufactured by Toyobo) according to a conventional method. The determined base sequence and amino acid sequence are as shown in SEQ ID NOs: 3 and 4. The molecular weight of the protein determined from the amino acid sequence is about 53000, and GDH of Acinetobacter baumannii JCM6841
Almost coincided with the molecular weight of

Example 5: Construction of an expression vector capable of replicating in Pseudomonas spp. 5 μg of the plasmid DNA obtained in Example 3 was digested with the restriction enzyme BamH.
By digestion with I and XhoI (manufactured by Toyobo), DNA containing a 1.9 Kb fragment containing the GDH gene was isolated.
Isolated DNA and pTS1137 cut with BamHI and XhoI
And 1 μg of T4 DNA ligase at 16 ° C.
After reacting for 6 hours, the DNA was ligated. The ligated DNA was transformed using competent cells of Escherichia coli DH5α. A colony having a plasmid having the GDH gene was found in the obtained colonies, and the plasmid was named pGLD3.

Example 6: Construction of an expression vector capable of replicating in Acinetobacter bacteria 5 μg of the plasmid DNA obtained in Example 3 was digested with the restriction enzyme MboI.
The DNA was digested with I (manufactured by Toyobo Co., Ltd.) and a DNA containing a 1.9 Kb fragment containing the GDH gene was isolated. Isolated D
1 μg of pWH1266 cut with NA and MscI (Toyobo)
Were reacted with 1 unit of T4 DNA ligase at 16 ° C. for 16 hours to ligate DNA. The ligated DNA was transformed using competent cells of Escherichia coli DH5α. A colony having a plasmid having the GDH gene was found in the obtained colonies, and the plasmid was replaced with the plasmid.
It was named pGLD4.

Comparative Example 1 Construction of Expression Vector for Escherichia coli Host 5 μg of the plasmid DNA obtained in Example 3 was digested with the restriction enzyme MboI.
The DNA was digested with I (manufactured by Toyobo Co., Ltd.) and a DNA containing a 1.8 Kb fragment containing the GDH gene was isolated. Isolated D
NA and 1 μg of pBluescript KS (+) digested with EcoRV were reacted with 1 unit of T4 DNA ligase at 16 ° C. for 16 hours to ligate the DNA. The ligated DNA was transformed using competent cells of Escherichia coli JM109. A colony having a plasmid having the GDH gene was found in the obtained colonies, and the plasmid was replaced with pGLD.
Named 5

Comparative Example 2: Escherichia coli JM109 / pG
Production of GDH with PQQ as Prosthetic Group from LD5 Terrific broth 500 ml was dispensed into a 2 L flask, and
After autoclaving at 21 ° C. for 15 minutes, the mixture was allowed to cool, and then 0.5 ml of 50 mg / ml ampicillin, which was separately sterilized and filtered, was added. The culture medium was inoculated with 5 ml of a culture solution of Escherichia coli JM109 / pPGLD5 previously cultured with shaking at 30 ° C. for 24 hours in an LB medium, and cultured with aeration and agitation at 37 ° C. for 24 hours. The GDH activity at the end of the culture was about 0.34 U / ml. On the other hand, after adding 10 μmol of PQQ to the activity measurement reagent, the activity was measured to be 120 U / ml.

Example 7: Preparation of transformant of microorganism having PQQ-producing ability Pseudomonas putida TE3493 (produced by Microtechnical Laboratory 12298)
No.) in LBG medium (LB medium + 0.3% glycerol)
At 30 ° C for 16 hours, and centrifuged (12000 rpm).
for 10 minutes), 8 ml of ice-cooled 5 mM K-phosphate buffer (pH 7.0) containing 300 mM sucrose was added, and the cells were suspended.
The cells were collected by centrifugation again (12000 rpm, 10 minutes), and the cells were ice-cooled and contained 5 mM K-phosphate buffer (pH 7.0) containing 300 mM sucrose.
0.4 ml was added to suspend the cells.

[0070] The plasmid DN obtained in Example 5 was added to the suspension.
A (pGLD3) (0.5 μg) was added, and the cells were transformed by electroporation. From colonies grown on LB medium containing 50 μg / ml streptomycin, P
A clone having a soluble GDH activity having QQ as a prosthetic group was obtained.

Similarly, using the above pGLD3, Pseudomonas putida TN1126 strain, Pseudomonas aeruginosa PA
O1162 strain, Pseudomonas fluorescens IFO12568
A transformant was obtained from the strain.

Further, Acinetobacter calcoaceticus NCIB11517 was cultured in an LB medium at 30 ° C. for 16 hours.
The cells were collected by centrifugation (12000 rpm, 10 minutes), and 8 ml of ice-cooled sterilized water was added to the cells to suspend the cells. The cells were collected again by centrifugation (12000 rpm, 10 minutes), and 0.4 ml of 10% glycerol was added to the cells to suspend the cells.

To 50 μl of the suspension, 0.5 μg of the plasmid DNA (pGLD4) obtained in Example 6 was added, followed by transformation by electroporation. From the colonies grown on the LB medium containing 50 μg / ml of tetracycline, a clone having soluble GDH activity having PQQ as a prosthetic group was obtained.

Example 8: GD using the obtained transformant
Comparison of the expression levels of H activity Five types of the transformants obtained in Example 7, the transformants obtained in Comparative Example 2, and Acinetobacter calcoaceticus NCIB11517 as a control were used in 50 ml of Terrific broth.
(1.2% polypeptone, 2.4% yeast extract, 0.4
% NaCl, 17 mM KH ₂ PO ₄ , 72 mM K ₂ H
(PO ₄ , pH 7.0) at 30 ° C. for 24 hours, and the cells were collected by centrifugation (12,000 rpm, 3 minutes). The cells were suspended in a 50 mM Tris / HCl buffer containing 1 mM CaCl ₂ , pH 7.5, and then disrupted by ultrasonication. Centrifugation (12000 rpm, 5 minutes)
After preparing a crude enzyme solution from which cell residues have been removed by
H activity was measured. Table 1 shows the results.

Example 9 Pseudomonas putida TE3493
Production of GDH Using PQQ as Prosthetic Group from / pGLD3 500 ml of Terrific broth is dispensed into a 2 L flask, and
The mixture was autoclaved at 21 ° C. for 15 minutes, allowed to cool, and then sterile-filtered separately at 50 mg / ml streptomycin 0.5%.
ml was added. The medium is previously LB medium at 30 ° C., 2
5 ml of the culture solution of Pseudomonas putida TE3493 / pGLD3 obtained in Example 7 obtained by culturing with shaking for 4 hours was inoculated.
The cells were cultured at 7 ° C. for 48 hours with aeration and stirring. GDH at the end of culture
The activity was about 45 U / ml.

The above cells were collected by centrifugation, and
Suspended in phosphate buffer, pH 7.0. The cell suspension was crushed by a French press and then centrifuged to obtain a supernatant as a crude enzyme solution. The crude enzyme solution was subjected to a nucleic acid removal treatment with polyethyleneimine and an ammonium sulfate fractionation treatment, and then desalted by gel filtration with Sephadex G-25 (Pharmacia Biotech), CM Sepharose (Pharmacia Biotech), Phenyl Sepharose (Pharmacia Biotech). ) Separation and purification by column chromatography gave a purified enzyme preparation.

The GDH sample obtained by the above method showed a substantially single band by electrophoresis (SDS-PAGE), and the specific activity at this time was about 2100 U / mg-protein.

The properties of GDH having PQQ obtained by the above method as a prosthetic group will be described below. Action: D-glucose + artificial electron acceptor → δ-
Gluconolactone + reduced electron acceptor Thermal stability: about 50 ° C. or less (treatment at pH 7.5 for 30 minutes) pH stability: 3.5 to 8.5 (treatment at 25 ° C. for 16 hours) Optimum temperature: about 40 ° C Optimum pH: 7.0 Molecular weight: 50,000

The above cells were collected by centrifugation,
It was suspended in a phosphate buffer (pH 7.0). The cell suspension was crushed by a French press and then centrifuged to obtain a supernatant as a crude enzyme solution. The crude enzyme solution was subjected to a nucleic acid removal treatment with polyethyleneimine and an ammonium sulfate fractionation treatment, and then desalted by gel filtration with Sephadex G-25 (Pharmacia Biotech), CM Sepharose (Pharmacia Biotech), Phenyl Sepharose (Pharmacia Biotech). ) Separation and purification by column chromatography to obtain a purified enzyme preparation.

The GDH preparation obtained by this method shows almost a single band by electrophoresis (SDS-PAGE),
The specific activity at this time was about 85 U / mg-protein. This enzyme was mixed with 1 mM CaCl ₂ and 10 μmol
After incubating at 37 ° C. for 1 hour in the presence of PQQ, specific activity was measured but was only about 460 U / mg-protein.

Example 10 Powderization of GDH The GDH of Example 9 dissolved in various buffers containing 1 mM CaCl ₂ and BSA (1.0 part by weight to 1.0 part by weight of GDH) was freeze-dried. Then, at 37 ° C.
The persistence of DH activity was measured. Table 2 shows the results.

[0082]

[Table 1]

[0083]

[Table 2]

[0084]

As described above, in the present invention, the PQ
Glucose dehydrogenase with Q as the prosthetic group was isolated. In addition, by genetic engineering technology utilizing a host-vector system using a microorganism capable of producing PQQ,
A method for producing GDH having QQ as a prosthetic group has been established. According to the production method of the present invention, it is possible to efficiently and inexpensively produce GDH having high-purity PQQ as a prosthetic group.

[0085]

[Sequence list]

SEQ ID NO: 1 Sequence length: 480 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Organism name: Acinetobacter calcoaceticus (Acin)
etobacter calcoaceticus) Strain name: NCIB11517 Sequence Met Asn Lys His Leu Leu Ala Lys Ile Thr Leu Leu Gly Ala Ala Gln 1 5 10 15 Leu Phe Thr Phe His Thr Ala Phe Ala Asp Ile Pro Leu Thr Pro Ala 20 25 30 Gln Phe Ala Lys Ala Lys Thr Glu Asn Phe Asp Lys Lys Val Ile Leu 35 40 45 Ser Asn Leu Asn Lys Pro His Ala Leu Leu Trp Gly Pro Asp Asn Gln 50 55 60 Ile Trp Leu Thr Glu Arg Ala Thr Gly Lys Ile Leu Arg Val Asn Pro 65 70 75 80 Val Ser Gly Ser Ala Lys Thr Val Phe Gln Val Pro Glu Ile Val Ser 85 90 95 Asp Ala Asp Gly Gln Asn Gly Leu Leu Gly Phe Ala Phe His Pro Asp 100 105 110 Phe Lys His Asn Pro Tyr Ile Tyr Ile Ser Gly Thr Phe Lys Asn Pro 115 120 125 Lys Ser Thr Asp Lys Glu Leu Pro Asn Gln Thr Ile Ile Arg Arg Tyr 130 135 140 Thr Tyr Asn Lys Thr Thr Asp Thr Phe Glu Lys Pro Ile Asp Leu Ile 145 150 155 160 Ala Gly Leu Pro Ser Ser Lys Asp His Gln Ser Gly Arg Leu Val Ile 165 170 175 Gly Pro Asp Gln Lys Ile Tyr Tyr Thr Ile Gly Asp Gln Gly Arg Asn 180 185 190 Gln Leu Ala Tyr Leu Phe Le u Pro Asn Gln Ala Gln His Thr Pro Thr 195 200 205 Gln Gln Glu Leu Asn Ser Lys Asp Tyr His Thr Tyr Met Gly Lys Val 210 215 220 Leu Arg Leu Asn Leu Asp Gly Ser Val Pro Lys Asp Asn Pro Ser Phe 225 230 235 240 Asn Gly Val Val Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro Gln 245 250 255 Gly Leu Ala Phe Ala Pro Asn Gly Lys Leu Leu Gln Ser Glu Gln Gly 260 265 270 Pro Asn Ser Asp Asp Glu Ile Asn Leu Val Leu Lys Gly Gly Asn Tyr 275 280 285 Gly Trp Pro Asn Val Ala Gly Tyr Lys Asp Asp Ser Gly Tyr Ala Tyr 290 295 300 Ala Asn Tyr Ser Ala Ala Thr Asn Lys Ser Gln Ile Lys Asp Leu Ala 305 310 315 320 Gln Asn Gly Ile Lys Val Ala Thr Gly Val Pro Val Thr Lys Glu Ser 325 330 335 Glu Trp Thr Gly Lys Asn Phe Val Pro Pro Leu Lys Thr Leu Tyr Thr 340 345 350 Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro Thr Cys Gly Glu Met Ala 355 360 365 Tyr Ile Cys Trp Pro Thr Val Ala Pro Ser Ser Ala Tyr Val Tyr Thr 370 375 380 Gly Gly Lys Lys Ala Ile Pro Gly Trp Glu Asn Thr Leu Leu Val Pro 385 390 395 400 400 Ser Leu Lys Arg Gly Val Il e Phe Arg Ile Lys Leu Asp Pro Thr Tyr 405 410 415 Ser Thr Thr Leu Asp Asp Ala Ile Pro Met Phe Lys Ser Asn Asn Arg 420 425 430 Tyr Arg Asp Val Ile Ala Ser Pro Glu Gly Asn Thr Leu Tyr Val Leu 435 440 445 Thr Asp Thr Ala Gly Asn Val Gln Lys Asp Asp Gly Ser Val Thr His 450 455 460 Thr Leu Glu Asn Pro Gly Ser Leu Ile Lys Phe Thr Tyr Asn Gly Lys 465 470 475 475 480

SEQ ID NO: 2 Sequence length: 1443 Sequence type: nucleic acid Topology: linear Sequence type: genomic DNA Origin Organism name: Acinetobacter calcoaceticus (Acin)
etobacter calcoaceticus) Strain name: NCIB11517 sequence ATG AAT AAA CAT TTA TTA GCA AAA ATC ACT CTT TTA GGT GCT GCA CAA 48 Met Asn Lys His Leu Leu Ala Lys Ile Thr Leu Leu Gly Ala Ala Gln 15 15 CTA TTT ACG TTT CAT ACG GCA TTT GCA GAT ATA CCT CTG ACA CCT GCT 96 Leu Phe Thr Phe His Thr Ala Phe Ala Asp Ile Pro Leu Thr Pro Ala 20 25 30 CAG TTC GCA AAA GCG AAA ACA GAA AAT TTT GAT AAA AAA GTG ATT CTG 144 Gln Phe Ala Lys Ala Lys Thr Glu Asn Phe Asp Lys Lys Val Ile Leu 35 40 45 TCC AAT TTA AAT AAA CCA CAT GCT TTG TTA TGG GGG CCA GAT AAT CAA 192 Ser Asn Leu Asn Lys Pro His Ala Leu Leu Trp Gly Pro Asp Asn Gln 50 55 60 ATT TGG TTA ACC GAA CGT GCA ACT GGC AAA ATT TTA AGA GTA AAT CCT 240 Ile Trp Leu Thr Glu Arg Ala Thr Gly Lys Ile Leu Arg Val Asn Pro 65 70 75 80 GTA TCT GGT AGC GCG AAA ACA GTA TTT CAG GTT CCT GAA ATT GTG AGT 288 Val Ser Gly Ser Ala Lys Thr Val Phe Gln Val Pro Glu Ile Val Ser 85 90 95 GAT GCT GAT GGG CAA AAT GGT TTG TTA GGT TTT GCT TTT CAT CCT GAC 336 Asp Ala Asp Gly Gln Asn Gly Leu Leu Gly Phe Ala Phe His Pro Asp 100 105 110 TTT AAA CAT AAC CCT TAT ATC TAT ATT TCA GGC ACT TTT AAA AAT CCA 384 Phe Lys His Asn Pro Tyr Ile Tyr Ile Ser Gly Thr Phe Lys Asn Pro 115 120 125 AAA TCT ACA GAT AAA GAG TTA CCT AAT CAG ACG ATT ATT CGT AGA TAT 432 Lys Ser Thr Asp Lys Glu Leu Pro Asn Gln Thr Ile Ile Arg Arg Tyr 130 135 140 ACC TAT AAT AAA ACT ACA GAT ACA TTT GAA AAG CCT ATT GAT TTG ATT 480 Thr Tyr Asn Lys Thr Thr Asp Thr Phe Glu Lys Pro Ile Asp Leu Ile 145 150 155 160 GCA GGT TTA CCG TCA TCA AAA GAT CAT CAG TCT GGT CGT CTC GTT ATT 528 Ala Gly Leu Pro Ser Ser Lys Asp His Gln Ser Gly Arg Leu Val Ile 165 170 175 GGT CCA GAC CAA AAA ATC TAC TAT ACG ATT GGT GAC CAA GGT CGT AAT 576 Gly Pro Asp Gln Lys Ile Tyr Tyr Thr Ile Gly Asp Gln Gly Arg Asn 180 185 190 CAG TTA GCT TAT CTG TTC TTA CCG AAT CAG GCA CAG CAT ACT CCG ACT 624 Gln Leu Ala Tyr Leu Phe Leu Pro Asn Gln Ala Gln His Thr Pro Thr 195 200 205 CAG CAA GAG CTC AAT AGT AAA GAC TAC CAT ACA TAT ATG GGT AAA GTA 672 Gln Gln Glu Leu Asn Ser Lys Asp Tyr His Thr Tyr Met Gly Lys Val 210 215 220 TTA CGC TTA AAT CTG GAC GGC AGT GTA CCT AAA GAC AAC CCA AGC TTT 720 Leu Arg Leu Asn Leu Asp Gly Ser Val Pro Lys Asp Asn Pro Ser Phe 225 230 235 240 AAC GGC GTA GTG AGT CAT ATC TAC ACT TTA GGG CAC CGT AAT CCA CAA 768 Asn Gly Val Val Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro Gln 245 250 255 GGT TTA GCA TTT GCC CCA AAT GGA AAG CTT TTA CAA TCT GAG CAA GGA 816 Gly Leu Ala Phe Ala Pro Asn Gly Lys Leu Leu Gln Ser Glu Gln Gly 260 265 270 CCA AAT TCT GAT GAT GAA ATT AAC CTT GTA TTA AAA GGT GGT AAC TAT 864 Pro Asn Ser Asp Asp Glu Ile Asn Leu Val Leu Lys Gly Gly Asn Tyr 275 280 285 GGC TGG CCA AAT GTA GCT GGT TAT AAA GAT GAC AGT GGT TAT GCC TAT 912 Gly Trp Pro Asn Val Ala Gly Tyr Lys Asp Asp Ser Gly Tyr Ala Tyr 290 295 300 GCA AAC TAT TCG GCA GCA ACC AAT AAA TCA CAA ATT AAA GAT TTA GCT 960 Ala Asn Tyr Ser Ala Ala Thr Asn Lys Ser Gln Ile Lys Asp Leu Ala 305 310 315 320 CAA AAC GGG ATA AAA GTA GCA ACA GGT GTT CCT GTG ACT AAA GAG TCT 1008 Gln Asn Gly Ile Lys Val Ala Thr Gly Val Pro Val Thr Lys Glu Ser 325 330 335 GAA TGG ACT GGT AAA AAC TTT GTG CCG CCT TTG AAA ACT TTA TAT ACG 1056 Glu Trp Thr Gly Lys Asn Phe Val Pro Pro Leu Lys Thr Leu Tyr Thr 340 345 350 GTA CAA GAT ACC TAT AAC TAT AAT GAC CCT ACT TGT GGT GAG ATG GCA 1104 Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro Thr Cys Gly Glu Met Ala 355 360 365 TAT ATT TGC TGG CCA ACG GTT GCA CCG TCA TCA GCA TAT GTA TAT ACG 1152 Tyr Ile Cys Trp Pro Thr Val Ala Pro Ser Ser Ala Tyr Val Tyr Thr 370 375 380 GGA GGC AAA AAA GCG ATT CCA GGG TGG GAA AAT ACA TTA TTG GTC CCA 1200 Gly Gly Lys Lys Ala Ile Pro Gly Trp Glu Asn Thr Leu Leu Val Pro 385 390 395 400 TCT TTA AAA CGT GGG GTG ATT TTC CGT ATT AAA TTG GAC CCG ACA TAT 1248 Ser Leu Lys Arg Gly Val Ile Phe Arg Ile Lys Leu Asp Pro Thr Tyr 405 410 415 AGC ACG ACT TTG GAT GAT GCT ATC CCA ATG TTT AAA AGC AAT AAC CGT 1296 Ser Thr Thr Leu Asp Asp Ala Ile Pro Met Phe Lys Ser Asn Asn Arg 420 425 430 TAT CGT GAT GTC ATC GCT AGT CCA G AA GGT AAT ACC TTA TAT GTG CTG 1344 Tyr Arg Asp Val Ile Ala Ser Pro Glu Gly Asn Thr Leu Tyr Val Leu 435 440 445 ACT GAT ACA GCG GGG AAT GTA CAA AAA GAT GAT GGT TCT GTC ACT CAT 1392 Thr Asp Thr Ala Gly Asn Val Gln Lys Asp Asp Gly Ser Val Thr His 450 450 455 460 ACT TTA GAG AAT CCC GGT TCT CTC ATT AAA TTT ACA TAT AAC GGT AAG 1440 Thr Leu Glu Asn Pro Gly Ser Leu Ile Lys Phe Thr Tyr Asn Gly Lys 465 470 475 480 TAA 1443

SEQ ID NO: 3 Sequence length: 480 Sequence type: amino acid Topology: linear Sequence type: protein Origin Organism name: Acinetobacte
r baumannii) Strain name: JCM6841 sequence Met Asn Lys His Leu Leu Ala Lys Ile Thr Leu Leu Gly Ala Ala Gln 1 5 10 15 Leu Phe Thr Phe His Thr Ala Phe Ala Asp Ile Pro Leu Thr Pro Ala 20 25 30 Gln Phe Ala Lys Ala Lys Thr Glu Asn Phe Asp Lys Lys Val Ile Leu 35 40 45 Ser Asn Leu Asn Lys Pro His Ala Leu Leu Trp Gly Pro Asp Asn Gln 50 55 60 Ile Trp Leu Thr Glu Arg Ala Thr Gly Lys Ile Leu Arg Val Asn Pro 65 70 75 80 Val Ser Gly Ser Ala Lys Thr Val Phe Gln Val Pro Glu Ile Val Ser 85 90 95 Asp Ala Asp Gly Gln Asn Gly Leu Leu Gly Phe Ala Phe His Pro Asp 100 105 110 Phe Lys His Asn Pro Tyr Ile Tyr Ile Ser Gly Thr Phe Lys Asn Pro 115 120 125 Lys Ser Thr Asp Lys Glu Leu Pro Asn Gln Thr Ile Ile Arg Arg Tyr 130 135 140 Thr Tyr Asn Lys Thr Thr Asp Thr Phe Glu Lys Pro Ile Asp Leu Ile 145 150 155 160 Ala Gly Leu Pro Ser Ser Lys Asp His Gln Ser Gly Arg Leu Val Ile 165 170 175 Gly Pro Asp Gln Lys Ile Tyr Tyr Thr Ile Gly Asp Gln Gly Arg Asn 180 185 190 Gln Leu Ala Tyr Leu Phe Leu Ser Asn Gln Ala Gln His Thr Pro Thr 195 200 205 Gln Gln Glu Leu Asn Ser Lys Asp Tyr His Thr Tyr Met Gly Lys Val 210 215 220 Leu Arg Leu Asn Leu Asp Gly Ser Ile Pro Lys Asp Asn Pro Ser Phe 225 230 235 240 Asn Gly Val Val Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro Gln 245 250 255 Gly Leu Ala Phe Ala Pro Asn Gly Lys Leu Leu Gln Ser Glu Gln Gly 260 265 270 Pro Asn Ser Asp Asp Glu Ile Asn Leu Val Leu Lys Gly Gly Asn Tyr 275 280 285 Gly Trp Pro Asn Val Ala Gly Tyr Lys Asp Asp Ser Gly Tyr Ala Tyr 290 295 300 Ala Asn Tyr Ser Ala Ala Thr Asn Lys Ser Gln Ile Lys Asp Leu Ala 305 310 310 315 320 Gln Asn Gly Ile Lys Val Ala Thr Gly Val Pro Val Thr Lys Glu Ser 325 330 335 Glu Trp Thr Gly Lys Asn Phe Val Pro Pro Leu Lys Thr Leu Tyr Thr 340 345 350 Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro Thr Cys Gly Glu Met Ala 355 360 365 Tyr Ile Cys Trp Pro Thr Val Ala Pro Ser Ser Ala Tyr Val Tyr Thr 370 375 380 Gly Gly Lys Lys Ala Ile Pro Gly Trp Glu Asn Thr Leu Leu Val Pro 385 390 395 400 Ser Leu Lys Arg Gly Val Ile Phe Arg Ile Lys Leu Asp Pro Thr Tyr 405 410 415 Ser Thr Thr Leu Asp Asp Ala Ile Pro Met Phe Lys Ser Asn Asn Arg 420 425 430 Tyr Arg Asp Val Ile Ala Ser Pro Glu Gly Gly Asn Thr Leu Tyr Val Leu 435 440 445 445 Thr Asp Thr Ala Gly Asn Val Gln Lys Asp Asp Gly Ser Val Thr His 450 455 460 Thr Leu Glu Asn Pro Gly Ser Leu Ile Lys Phe Thr Tyr Asn Gly Lys 465 470 475 480

SEQ ID NO: 4 Sequence length: 1443 Sequence type: nucleic acid Topology: linear Sequence type: genomic DNA Origin Organism name: Acinetobacte
r baumannii) Strain name: JCM6841 sequence ATG AAT AAA CAT TTA TTA GCA AAA ATC ACT CTT TTA GGT GCT GCA CAA 48 Met Asn Lys His Leu Leu Ala Lys Ile Thr Leu Leu Gly Ala Ala Gln 1 5 10 15 CTA TTT ACG TTT CAT ACG GCA TTT GCA GAT ATA CCT CTG ACA CCT GCT 96 Leu Phe Thr Phe His Thr Ala Phe Ala Asp Ile Pro Leu Thr Pro Ala 20 25 30 CAG TTC GCA AAA GCG AAA ACA GAA AAT TTT GAT AAA AAA GTG ATT CTG 144 Gln Phe Ala Lys Ala Lys Thr Glu Asn Phe Asp Lys Lys Val Ile Leu 35 40 45 TCC AAT TTA AAT AAA CCA CAT GCT TTG TTA TGG GGG CCA GAT AAT CAA 192 Ser Asn Leu Asn Lys Pro His Ala Leu Leu Trp Gly Pro Asp Asn Gln 50 55 60 ATT TGG TTA ACC GAA CGT GCA ACT GGC AAA ATT TTA AGA GTA AAT CCT 240 Ile Trp Leu Thr Glu Arg Ala Thr Gly Lys Ile Leu Arg Val Asn Pro 65 70 75 80 GTA TCT GGT AGC GCG AAA ACA GTA TTT CAG GTT CCT GAA ATT GTG AGT 288 Val Ser Gly Ser Ala Lys Thr Val Phe Gln Val Pro Glu Ile Val Ser 85 90 95 GAT GCT GAT GGG CAA AAt GGT TTG TTA GGT TTT GCT TTT CAT CCT GAC 336 Asp Ala Asp Gly Gln Asn Gly Le u Leu Gly Phe Ala Phe His Pro Asp 100 105 110 TTT AAA CAT AAC CCT TAT ATC TAT ATT TCA GGC ACT TTT AAA AAT CCA 384 Phe Lys His Asn Pro Tyr Ile Tyr Ile Ser Gly Thr Phe Lys Asn Pro 115 120 125 AAA TCT ACA GAT AAA GAG TTA CCT AAT CAG ACA ATT ATT CGT AGA TAT 432 Lys Ser Thr Asp Lys Glu Leu Pro Asn Gln Thr Ile Ile Arg Arg Tyr 130 135 140 ACC TAT AAT AAA ACT ACA GAT ACA TTT GAA AAG CCT ATT GAT TTG ATT 480 Thr Tyr Asn Lys Thr Thr Asp Thr Phe Glu Lys Pro Ile Asp Leu Ile 145 150 155 160 GCA GGT TTA CCG TCA TCA AAA GAT CAT CAG TCT GGT CGT CTC GTT ATT 528 Ala Gly Leu Pro Ser Ser Lys Asp His Gln Ser Gly Arg Leu Val Ile 165 170 175 GGT CCA GAC CAA AAA ATC TAC TAT ACG ATT GGT GAC CAA GGT CGT AAT 576 Gly Pro Asp Gln Lys Ile Tyr Tyr Thr Ile Gly Asp Gln Gly Arg Asn 180 185 190 CAG TTA GCT TAT CTA TTC TTA TCG AAT CAG GCA CAG CAT ACT CCG ACT 624 Gln Leu Ala Tyr Leu Phe Leu Ser Asn Gln Ala Gln His Thr Pro Thr 195 200 205 CAG CAA GAG CTC AAT AGT AAA GAC TAC CAT ACA TAT ATG GGT AAA GTA 672 Gln Gln Glu Leu As n Ser Lys Asp Tyr His Thr Tyr Met Gly Lys Val 210 215 220 TTA CGC TTA AAT CTG GAC GGC AGT ATA CCT AAA GAC AAC CCA AGC TTT 720 Leu Arg Leu Asn Leu Asp Gly Ser Ile Pro Lys Asp Asn Pro Ser Phe 225 230 235 240 AAC GGC GTA GTG AGT CAT ATC TAC ACT TTA GGG CAC CGT AAT CCA CAA 768 Asn Gly Val Val Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro Gln 245 250 255 GGT TTA GCA TTT GCC CCA AAT GGA AAG CTT TTA CAA TCT GAG CAA GGG 816 Gly Leu Ala Phe Ala Pro Asn Gly Lys Leu Leu Gln Ser Glu Gln Gly 260 265 270 CCA AAT TCT GAT GAT GAA ATT AAC CTT GTA TTA AAA GGT GGT AAC TAT 864 Pro Asn Ser Asp Asp Glu Ile Asn Leu Val Leu Lys Gly Gly Asn Tyr 275 280 285 285 GGC TGG CCA AAT GTA GCT GGT TAT AAA GAT GAC AGT GGT TAT GCC TAT 912 Gly Trp Pro Asn Val Ala Gly Tyr Lys Asp Asp Ser Gly Tyr Ala Tyr 290 295 300 GCA AAC TAT TCG GCA GCA ACC AAT AAA TCA CAA ATT AAA GAT TTA GCT 960 Ala Asn Tyr Ser Ala Ala Thr Asn Lys Ser Gln Ile Lys Asp Leu Ala 305 310 315 320 CAA AAC GGG ATA AAA GTA GCA ACA GGT GTT CCT GTG ACT AAA GAG TCT 1008 G ln Asn Gly Ile Lys Val Ala Thr Gly Val Pro Val Thr Lys Glu Ser 325 330 335 GAA TGG ACT GGT AAA AAC TTT GTG CCA CCT TTG AAA ACT TTA TAT ACG 1056 Glu Trp Thr Gly Lys Asn Phe Val Pro Pro Leu Lys Thr Leu Tyr Thr 340 345 350 GTA CAA GAT ACC TAT AAC TAT AAT GAC CCT ACT TGT GGT GAG ATG GCA 1104 Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro Thr Cys Gly Glu Met Ala 355 360 365 TAT ATT TGC TGG CCA ACG GTT GCA CCG TCA TCG GCA TAT GTA TAT ACG 1152 Tyr Ile Cys Trp Pro Thr Val Ala Pro Ser Ser Ala Tyr Val Tyr Thr 370 375 380 GGA GGC AAA AAA GCG ATT CCA GGG TGG GAA AAT ACA TTA TTG GTC CCA 1200 Gly Gly Lys Lys Ala Ile Pro Gly Trp Glu Asn Thr Leu Leu Val Pro 385 390 395 400 TCT TTA AAA CGT GGG GTG ATT TTC CGT ATT AAA TTG GAC CCG ACA TAT 1248 Ser Leu Lys Arg Gly Val Ile Phe Arg Ile Lys Leu Asp Pro Thr Tyr 405 410 415 AGC ACG ACT TTG GAT GAT GCT ATC CCA ATG TTT AAA AGC AAT AAC CGT 1296 Ser Thr Thr Leu Asp Asp Ala Ile Pro Met Phe Lys Ser Asn Asn Arg 420 425 430 TAT CGT GAT GTC ATC GCT AGT CCA GAA GGT AAT ACC TTA TAT GTG CTG 1344 Tyr Arg Asp Val Ile Ala Ser Pro Glu Gly Asn Thr Leu Tyr Val Leu 435 440 445 ACT GAT ACA GCG GGA AAT GTA CAA AAA GAT GAT GGT TCA GTC ACT CAT 1392 Thr Asp Thr Ala Gly Asn Val Gln Lys Asp Asp Gly Ser Val Thr His 450 455 460 ACT TTA GAG AAT CCC GGT TCT CTC ATT AAA TTT ACA TAT AAC GGT AAG 1440 Thr Leu Glu Asn Pro Gly Ser Leu Ile Lys Phe Thr Tyr Asn Gly Lys 465 470 475 475 480 TAA 1443

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1:19) (C12N 1/21 C12R 1:19) (C12N 15/09 ZNA C12R 1:01) (72) Inventor Adachi Tosei 2-2-204 Shibazaki-cho, Yamaguchi-shi, Yamaguchi Prefecture (72) Inventor Kazunobu Matsushita 2645-27, Yoshikiki, Yamaguchi-shi

Claims (34)

[Claims] 1. A glucose dehydrogenase comprising PQQ which is a protein of the following (a) or (b) as a prosthetic group: (A) a protein consisting of the amino acid sequence described in Sequence Listing / SEQ ID NO: 1 (b) an amino acid sequence (a) comprising one or several amino acid sequences deleted, substituted or added, and Protein having glucose dehydrogenase activity 2. A glucose dehydrogenase having PQQ, which is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 as a prosthetic group. 3. A glucose dehydrogenase comprising PQQ which is a protein of the following (e) or (f) as a prosthetic group: (E) a protein comprising the amino acid sequence described in SEQ ID NO: 3 (f) an amino acid sequence (e) wherein one or several amino acid sequences are deleted, substituted or added, and Protein having glucose dehydrogenase activity 4. A glucose dehydrogenase comprising PQQ, which is a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 as a prosthetic group. 5. A gene encoding glucose dehydrogenase having PQQ which is a protein of the following (a) or (b) as a prosthetic group: (A) a protein consisting of the amino acid sequence described in Sequence Listing / SEQ ID NO: 1 (b) an amino acid sequence (a) comprising one or several amino acid sequences deleted, substituted or added, and Protein having glucose dehydrogenase activity 6. A gene encoding glucose dehydrogenase having PQQ which is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 as a prosthetic group. 7. A gene encoding glucose dehydrogenase having PQQ, a protein of the following (c) or (d), as a prosthetic group: (C) DNA consisting of the nucleotide sequence described in SEQ ID NO: 2 (d) In the above sequence (c), one or several bases are deleted, substituted or added, and glucose dehydrogenase activity DNA encoding a protein having 8. A gene encoding glucose dehydrogenase having PQQ having a DNA consisting of the nucleotide sequence of SEQ ID NO: 2 as a prosthetic group. 9. A gene encoding glucose dehydrogenase having PQQ, a protein of the following (e) or (f), as a prosthetic group: (E) a protein comprising the amino acid sequence described in SEQ ID NO: 3 (f) an amino acid sequence (e) wherein one or several amino acid sequences are deleted, substituted or added, and Protein having glucose dehydrogenase activity 10. A gene encoding glucose dehydrogenase having PQQ which is a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 as a prosthetic group. 11. A gene encoding glucose dehydrogenase having PQQ, which is a protein of the following (g) or (h), as a prosthetic group: (G) DNA consisting of the base sequence described in SEQ ID NO: 4 (h) In the above sequence (g), one or several bases are deleted, substituted or added, and glucose dehydrogenase activity DNA encoding a protein having 12. A gene encoding glucose dehydrogenase having PQQ having a DNA consisting of the nucleotide sequence of SEQ ID NO: 4 as a prosthetic group. 13. The P according to claim 5, wherein
A recombinant vector containing a gene encoding glucose dehydrogenase having QQ as a prosthetic group. 14. The recombinant vector according to claim 13, wherein a DNA fragment containing a gene encoding glucose dehydrogenase having PQQ as a prosthetic group is integrated and can be replicated in a microorganism capable of producing PQQ. 15. A transformant obtained by transforming a microorganism capable of producing PQQ with the recombinant vector according to claim 13 or 14. 16. A glucose dehydrogenase having PQQ as a prosthetic group is Acinetobacter calcoaceticus or Acinetobacter baumannii.
The transformant according to claim 15, which is derived. 17. The microorganism capable of producing PQQ is a microorganism belonging to the genus Pseudomonas or the genus Acinetobacter.
The transformant as described above. 18. The microorganism having PQQ-producing ability may be Pseudomonas aeruginosa or Pseudomonas fluorescens.
scens), Pseudomonas puti
da), Acinetobacter calcoaceticus (Acinet
The transformant according to claim 15, which is a microorganism selected from the group consisting of A. bacterium calcoaceticus) and Acinetobacter baumannii. 19. The transformant according to claim 17, wherein the microorganism capable of producing PQQ is Pseudomonas putida. 20. A microorganism capable of producing PQQ is Acinetobacter calcoaceticas.
18. The transformant according to claim 17, which is Oaceticus) or Acinetobacter baumannii. 21. The glucose dehydrogenase having PQQ as a prosthetic group is soluble.
The transformant according to item 1. 22. A transformant obtained by transforming a microorganism capable of producing PQQ with a recombinant vector comprising a DNA fragment containing a gene encoding glucose dehydrogenase having PQQ as a prosthetic group, A method for producing glucose dehydrogenase, comprising collecting glucose dehydrogenase having PQQ as a prosthetic group from the culture. 23. A glucose dehydrogenase having PQQ as a prosthetic group is Acinetobacter calcoaceticus or Acinetobacter baumannii.
The method for producing glucose dehydrogenase according to claim 22, which is derived from a microorganism. 24. The microorganism having PQQ-producing ability is a microorganism belonging to the genus Pseudomonas or the genus Acinetobacter.
A method for producing the glucose dehydrogenase described above. 25. A microorganism capable of producing PQQ is Pseudomonas aeruginosa, Pseudomonas fluorescens.
scens), Pseudomonas puti
da), Acinetobacter calcoaceticus (Acinet
24. The method for producing glucose dehydrogenase according to claim 23, wherein the microorganism is a microorganism selected from the group consisting of A. bacterium calcoaceticus) and Acinetobacter baumannii. 26. The method for producing glucose dehydrogenase according to claim 23, wherein the microorganism having PQQ producing ability is Pseudomonas putida. 27. The microorganism capable of producing PQQ is Acinetobacter calcoaceticas.
24. The method for producing glucose dehydrogenase according to claim 23, which is Oaceticus) or Acinetobacter baumannii. 28. The glucose dehydrogenase having PQQ as a prosthetic group is soluble.
3. The method for producing glucose dehydrogenase according to 1.). 29. A method for stabilizing glucose dehydrogenase, comprising freeze-drying and maintaining glucose dehydrogenase having PQQ as a prosthetic group in the presence of a GOOD buffer. 30. The method for stabilizing glucose dehydrogenase according to claim 29, wherein calcium coexists. 31. The GOOD buffer is PIPES, ME
32. The method for stabilizing glucose dehydrogenase according to claim 30 or 31, which is a buffer selected from the group consisting of S and MOPS. 32. A stabilized glucose dehydrogenase composition, wherein glucose dehydrogenase having PQQ as a prosthetic group is freeze-dried and maintained in the presence of a GOOD buffer. 33. The glucose dehydrogenase composition according to claim 32, wherein calcium is present. 34. The GOOD buffer is PIPES, ME
The glucose dehydrogenase composition according to claim 32 or 33, which is a buffer selected from the group consisting of S and MOPS.