JP2001346587A - Glucose dehydrogenase excellent in substrate specificity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a water-soluble PQQGDH(pyrroloquinolinequinone) (glucose dehydrogenase) having a reactivity to lactose or maltose lower than that to glucose. SOLUTION: This PQQGDH has an amino acid residue substituted from an amino acid residue corresponding to 167 aspartic acid residue of water-soluble PQQGDH derived from Acinetobacter calcoaceticus.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a glucose dehydrogenase (G) having pyrroloquinoline quinone (PQQ) as a coenzyme.
DH), wherein the specific amino acid residue is replaced with another amino acid residue. The modified PQQGDH of the present invention is useful for glucose quantification in clinical tests, food analysis, and the like.

[0002]

2. Description of the Related Art PQQGDH is a glucose dehydrogenase using pyrroloquinoline quinone as a coenzyme, and catalyzes a reaction of oxidizing glucose to produce gluconolactone.

[0003] It is known that PQQGDH includes a membrane-bound enzyme and a water-soluble enzyme. Membrane-bound PQQGDH
Is a single peptide protein having a molecular weight of about 87 kDa, and is widely found in various Gram-negative bacteria. For example, AM. Cleton-Jansen
t al. , J. et al. Bacteriol. (199
0) 172, 6308-6315.
On the other hand, water-soluble PQQGDH is Acinetobacter calcoaceti
Its presence has been confirmed in several strains of C. cus (Biosci. Biotech. Biochem. (1995), 59 (8), 1548-15).
55), and its structural gene has been cloned and its amino acid sequence has been determined (Mol. Gen. Genet. (1989), 217:
430-436). A. calcoaceticus-derived water-soluble PQQGDH
Is a homodimer with a molecular weight of about 50 kDa. Other P
The QQ enzyme has almost no homology on the primary structure of the protein.

[0004] Recently, the results of X-ray crystal structure analysis of the present enzyme have been reported, and the higher order structure of the present enzyme including the active center has been revealed. (A. Oubrie et al., J. Mol. Biol., 28
9, 319-333 (1999); A. Oubrie et al., The EMBO Journ
al, 18 (19) 5187-5194 (1999); A. Oubrie et al. PNA
S, 96 (21), 11787-11791 (1999)). According to these articles, it was revealed that water-soluble PQQGDH is a β-propeller protein composed of six W-motifs.

[0005] Blood glucose concentration is an extremely important indicator for clinical diagnosis as an important marker of diabetes. In addition, quantification of glucose concentration in fermentation production using microorganisms is an important item in process monitoring. Conventionally, glucose is glucose oxidase (GOD) or glucose 6-phosphate dehydrogenase (G
6PDH) by the enzymatic method. However, in the method using GOD, it was necessary to add catalase or peroxidase to the assay system in order to quantify hydrogen peroxide generated during the glucose oxidation reaction. Biosensors using GOD have also been developed, but the reaction depends on the concentration of dissolved oxygen in the aqueous solution, making it unsuitable for high-concentration glucose samples. There was a possibility. On the other hand, G6PDH has been used for glucose quantification based on a spectroscopic technique, but has the trouble that NAD (P) as a coenzyme must be added to the reaction system. Therefore, application of PQQGDH has attracted attention as a new enzyme that replaces the enzyme used in the conventional glucose enzyme quantification method. PQQGDH
Has a high oxidizing activity on glucose,
Since PQQGDH is a coenzyme-linked enzyme and does not require oxygen as an electron acceptor, it is expected to be applied to assay fields such as glucose sensor recognition elements. However, PQQGDH had a problem of low selectivity for glucose.

[0006]

Accordingly, the present invention provides a water soluble PQ having improved selectivity for glucose.
It aims to provide QGDH. The present invention, in particular,
An object of the present invention is to provide a water-soluble PQQGDH having a lower reactivity to lactose or maltose as compared with a reactivity to glucose in order to increase the sensitivity of measuring blood glucose concentration.

[0007]

Means for Solving the Problems The present inventor has improved the conventional water-soluble PQQGDH to increase its selectivity for glucose, and has modified P-QQGDH applicable to clinical tests and food analysis.
As a result of intensive studies to develop QQGDH, an enzyme having extremely high selectivity for glucose was successfully obtained by introducing an amino acid mutation in a specific region of water-soluble PQQGDH. That is, the present invention relates to a water-soluble PQQG derived from Acinetobacter calcoaceticus.
PQQG wherein the amino acid residue corresponding to the aspartic acid residue at position 167 of DH is substituted with another amino acid residue
Provide DH. The PQQGDH of the present invention is characterized by having improved selectivity for glucose as compared with natural water-soluble PQQGDH. Preferably, the modified PQQGDH of the present invention has a lower reactivity to lactose or maltose than to the wild type as compared to the reactivity to glucose. More preferably, when the reactivity to glucose is 100%, the activity for lactose or maltose is 50% or less, more preferably 40% or less, and further preferably 30% or less.

[0008] As used herein with respect to amino acid residues or regions, the term "corresponding" refers to the function of a given amino acid residue or region in two or more proteins that are structurally similar but not identical to each other. To have For example, in water-soluble PQQGDH derived from organisms other than Acinetobacter calcoaceticus, Acinetobacte
It is assumed that there is a region having a high amino acid sequence similarity to the region from residues 164 to 170 of water-soluble PQQGDH derived from R. calcoaceticus, and that the region plays the same role in the protein in view of the secondary structure of the protein. If reasonably conceivable, the region may be "Acinetobacter calcoaceticu
s-derived water-soluble PQQGDH, corresponding to the region from residue 164 to residue 170 ". Further, the fourth of the region
The second amino acid residue is `` Acinetobacter calcoaceticus
It corresponds to residue 167 of the derived water-soluble PQQGDH. " In the present specification, amino acid positions are numbered with starting methionine as 1.

Preferably, in the PQQGDH of the present invention, the aspartic acid residue at position 167 of the amino acid sequence represented by SEQ ID NO: 1 is substituted with another amino acid residue.

In another aspect, the present invention provides a PQQ
GDH comprises the sequence: Ser Gln His Xaa Lys Ser Ser, where Xaa is any natural amino acid residue other than Asp.

The present invention also provides a gene encoding the above-mentioned PQQGDH, a vector containing the gene, a transformant containing the gene, a method for producing the PQQGDH of the present invention, a glucose assay kit containing the PQQGDH of the present invention, Provide a glucose sensor.

Since the enzyme protein of PQQGDH of the present invention has high selectivity for glucose, it can be applied to highly sensitive and highly selective measurement of glucose.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Structure of Modified PQQGDH Mutations are randomly introduced into the coding region of a gene encoding water-soluble PQQGDH by error-prone PCR, and a water-soluble PQ having mutations in amino acid residues is introduced.
A library of QGDH was constructed. This was transformed into Escherichia coli and screened for PQQGDH activity, and the PQ activity at 100 mM glucose was equivalent to wild-type PQQGDH, but the activity at 100 mM maltose was lower than wild-type PQQGDH.
A number of clones expressing QGDH were obtained.

Analysis of the gene sequence of one of these clones revealed that the 167th Asp was replaced by Glu. Further, when this residue was replaced with glycine, histidine, tyrosine, alanine, lysine, asparagine, glutamine, valine, cysteine, serine or tryptophan residue, any of the residues could be replaced by glucose rather than wild-type water-soluble PQQGDH. A mutant enzyme with improved selectivity for was obtained.

In the modified PQQGDH of the present invention,
As long as it has the desired glucose dehydrogenase activity,
Further, a part of another amino acid residue may be deleted or substituted, or another amino acid residue may be added. Various methods for such site-specific nucleotide sequence substitution are known in the art, for example,
Sambrook et al., “Molecular Cloning; A Laboratory Manua
l ”, 2nd edition, 1989, Cold Spring Harbor Laboratory Pre
ss, New York.

Furthermore, those skilled in the art will also compare the primary structure of water-soluble PQQGDH derived from another bacterium in parallel with the primary structure of the protein or the secondary structure predicted based on the primary structure of the enzyme. By comparison, Acinetob
The first of water-soluble PQQGDH derived from acter calcoaceticus
A modified PQQGDH having improved selectivity for glucose by easily recognizing the residue corresponding to the 67th aspartic acid, and replacing such amino acid residue with another amino acid residue according to the present invention. Can be obtained. These modified PQQGDHs are also within the scope of the present invention. Process for producing modified PQQGDH Natural water-soluble PQ derived from Acinetobacter calcoaceticus
The sequence of the gene encoding QGDH is defined in SEQ ID NO: 2.

In the gene encoding the modified PQQGDH of the present invention, the nucleotide sequence encoding the desired amino acid residue in the gene encoding the natural water-soluble PQQGDH is replaced with the nucleotide sequence encoding the amino acid residue to be mutated. It can be constructed by substitution.

The mutant gene thus obtained is inserted into a vector for gene expression (eg, a plasmid), and transformed into a suitable host (eg, Escherichia coli). Many vector-host systems for expressing foreign proteins are known in the art, and hosts include, for example,
Various things such as bacteria, yeast, and cultured cells can be used.

When introducing a random mutation, a mutation is randomly introduced into a target region by an error-prone PCR method, and a mutant water-soluble PQQGDH gene library having a mutation introduced into the region is constructed.

This was transformed into Escherichia coli, and PQQGDH
Each clone is screened for its selectivity for glucose. Since water-soluble PQQGDH is secreted into the periplasmic space when expressed in E. coli,
Enzyme activity can be easily assayed using the cells themselves. Using this library as a dye, PMS-DC
The activity of PQQGDH was visually determined by adding IP, and the activity of glucose at 100 mM concentration was equivalent to that of wild-type PQQGDH, but the activity of 100 mM maltose was lower than that of wild-type PQQGDH.
A clone expressing GDH is selected, and the gene sequence is analyzed to confirm the mutation.

The modified PQQ obtained as described above
After the transformant expressing GDH is cultured and the cells are collected from the culture by centrifugation or the like, the cells are disrupted by a French press or the like, or the periplasmic enzyme is released into the medium by osmotic shock. This is ultracentrifuged to obtain a water-soluble fraction containing PQQGDH. Alternatively, the expressed PQQGDH can be secreted into the culture medium by using an appropriate host vector system. The obtained water-soluble fraction was subjected to ion exchange chromatography, affinity chromatography, H
The purified PQQGDH of the present invention is prepared by purification by PLC or the like. Method for Measuring Enzyme Activity The PQQGDH of the present invention has a function of catalyzing a reaction of oxidizing glucose to produce gluconolactone using PQQ as a coenzyme.

The enzyme activity can be measured by quantifying the amount of PQQ reduced by the oxidation of glucose by PQQGDH by a color reaction of a redox dye. As the coloring reagent, for example, PMS (phenazine methosulfate) -DCIP (2,6-dichlorophenolindophenol), potassium ferricyanide, ferrocene and the like can be used. Selectivity evaluation method The selectivity of glucose of PQQGDH of the present invention is as follows:
As a substrate, enzyme activity was measured as described above using various sugars such as 2-deoxy-D-glucose, mannose, allose, 3-o-methyl-D-glucose, galactose, xylose, lactose and maltose, It can be evaluated by examining the relative activity to the activity when glucose is used as a substrate. Glucose Assay Kit The present invention also features a glucose assay kit comprising a modified PQQGDH according to the present invention. The glucose assay kit of the present invention comprises the modified PQQG according to the present invention.
DH is included in an amount sufficient for at least one assay. Typically, the kit contains, in addition to the modified PQQGDH of the present invention, buffers necessary for the assay, mediators, glucose standard solutions for preparing calibration curves, and guidelines for use. Modified P according to the invention
QQGDH can be provided in various forms, for example, as a lyophilized reagent or as a solution in a suitable storage solution. Preferably, the modified PQQGD of the present invention
Although H is provided in a holo-form, it can also be provided in the form of an apoenzyme and holo-formated at the time of use. Glucose Sensor The present invention also features a glucose sensor using the modified PQQGDH according to the present invention. As the electrode, a carbon electrode, a gold electrode, a platinum electrode or the like is used, and the enzyme of the present invention is immobilized on the electrode. Examples of the immobilization method include a method using a crosslinking reagent, a method of enclosing in a polymer matrix, a method of coating with a dialysis membrane, a photocrosslinkable polymer, a conductive polymer, and a redox polymer. It may be fixed in a polymer together with a representative electron mediator or adsorbed and fixed on an electrode, or may be used in combination. Preferably, the modified PQQGDH of the present invention is immobilized on the electrode in a holated form, but it can also be immobilized in the form of an apoenzyme, providing PQQ as a separate layer or in solution. Typically, the modified PQQGDH of the present invention is immobilized on a carbon electrode using glutaraldehyde, and then treated with a reagent having an amine group to block glutaraldehyde.

The measurement of the glucose concentration can be performed as follows. Put buffer solution in thermostat cell,
And CaCl ₂ , and a mediator are added and maintained at a constant temperature. As the mediator, potassium ferricyanide, phenazine methosulfate and the like can be used. The modified PQQ of the present invention as a working electrode
A GDH-immobilized electrode is used, and a counter electrode (for example, a platinum electrode) and a reference electrode (for example, an Ag / AgCl electrode) are used. After a constant voltage is applied to the carbon electrode and the current becomes steady, a sample containing glucose is added and the increase in the current is measured. The glucose concentration in the sample can be calculated according to a calibration curve prepared with a standard concentration glucose solution.

[0024]

EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Construction and Screening of Mutant PQQGDH Gene Library Plasmid pGB2 was inserted into the vector pTrc99A (manufactured by Pharmacia) at the multiple cloning site of Acinetob.
It is one into which a structural gene encoding PQQGDH derived from acter calcoaceticus has been inserted (FIG. 1). Using this plasmid as a template, mutations were randomly introduced into various regions by error-prone PCR. The PCR reaction was carried out in a solution having the composition shown in Table 1 at 94 ° C. for 3 minutes, then at 30 cycles of 94 ° C. for 3 minutes, 50 ° C. for 2 minutes, and 72 ° C. for 2 minutes, and finally for 10 minutes at 72 ° C. I went in.

[0026]

Table 1 Taq DNA polymerase (5 U / μl) 0.5 μl template DNA 1.0 μl forward primer ABF 4.0 μl reverse primer ABR 4.0 μl 10 × Taq polymerase buffer 10.0 μl 1M β-mercaptoethanol 1.0 μl DMSO 10. _{0μl 5mM MnCl 2 10.0μl 10mM dGTP 2.0μl} 2mM dATP 2.0μl 10mM dCTP 2.0μl 10mM dTTP 2.0μl H 2 O 51.5μl 100.0μl resulting transformed the library of mutated soluble PQQGDH in E. coli Converted and each formed colony was transferred to a microtiter plate. Replicate the colony to another plate, and add glucose concentration of 100 mM to one plate.
And PMS-DCIP and add 1 to the other plate
00 mM maltose and PMS-DCIP were added, and the activity of both PQQGDH was determined visually. Many clones were obtained in which the activity on maltose was significantly lower than the activity exhibited by glucose on the two plates.

One of the clones was arbitrarily selected and the gene sequence was analyzed. As a result, it was found that the aspartic acid at position 167 was mutated to glutamic acid.

Example 2 Construction of Modified PQQGDH Gene Based on the structural gene of PQQGDH derived from Acinetobacter calcoaceticus shown in SEQ ID NO: 2, the sequence: 5'-CC TGA CTG ATG GTG TTT TGA TGA AGG-3 '(SEQ ID NO: : 4) synthesizing the oligonucleotide target primer of
The aspartic acid at position 167 was replaced with glycine. The site-specific mutation was performed using the plasmid pGB2 according to the method shown in FIG.

The vector plasmid pKF18k (Takara Shuzo Co., Ltd.) was used for PQQG derived from Acinetobacter calcoaceticus.
A Kpn I-Hind III fragment containing a part of the gene encoding DH was incorporated and used as a template. 50 pmol of this template, 5 pmol of the selection primer attached to the Mutan (registered trademark) -Express Km kit manufactured by Takara Shuzo Co., Ltd. and 50 pmol of the phosphorylated target primer were mixed together with 1/10 of the total (20 μl) of the annealing buffer of the same kit. ,
The plasmid was denatured by heat treatment at 100 ° C for 3 minutes.
It became a main chain. The selection primer is for reversing the double amber mutation on the kanamycin resistance gene of pKF18k. This was placed on ice for 5 minutes to allow the primer to anneal. To this, 3 μl of the same kit extension buffer and 1 μl of T4
DNA ligase, 1 μl of T4 DNA polymerase and 5 μl of sterile water were added to synthesize a complementary strand.

This was transformed into E. coli BMH71-18mutS, which is a strain deficient in mismatch repair ability of DNA, and the plasmid was amplified by shaking culture overnight.

Next, the plasmid extracted therefrom was placed in E.co.
liMV1184, and plasmids were extracted from the colonies. These plasmids were sequenced to confirm the introduction of the desired mutation. This fragment was cloned into wild-type PQQ on plasmid pGB2.
The KDH I-Hind III fragment of the gene encoding GDH was replaced to construct a modified PQQGDH gene.

Similarly, Asp167Ala, Asp167His, Asp1
67Lys, Asp167Asn, Asp167Gln, Asp167Val, Asp167Ty
A modified PQQGDH gene having each mutation of r, Asp167Cys, Asp167Ser, and Asp167Trp was constructed.

Example 3 Preparation of Modified PQQGDH A gene encoding a wild-type or modified PQQGDH was pTrc99, an expression vector for E. coli
A (Pharmacia) was inserted into the multiple cloning site, and the constructed plasmid was transformed into E. coli DH5α strain. This was added to 450 ml of L medium (ampicillin 50 μg / ml, chloramphenicol 30 μg / ml).
Was shaken at 37 ° C. overnight using a Sakaguchi flask, and inoculated into 71 L medium containing 1 mM CaCl ₂ and 500 μMPQQ. About 3 hours after the start of the culture, isopropylthiogalactoside was added to a final concentration of 0.3 mM, and then the cells were cultured for 1.5 hours. The cells were recovered from the culture by centrifugation (5000 × g, 10 minutes, 4 ° C.), and the cells were washed twice with a 0.85% NaCl solution. The collected cells are crushed by a French press and centrifuged (10000 xg,
(15 minutes, 4 ° C.) to remove unbroken cells. Ultracentrifugation of the supernatant (160500 × g (40000 rpm), 90 minutes, 4 ° C.)
Thus, a water-soluble fraction was obtained. This was used in the following examples as a crude purified enzyme preparation.

Further, the water-soluble fraction thus obtained was
Dialyzed overnight against M phosphate buffer pH 7.0. TSKg packed column for cation exchange chromatography in which the dialyzed sample was equilibrated with 10 mM phosphate buffer pH 7.0.
el CM-TOYOPEARL 650M (Tosoh Corporation). The column was washed with 750 ml of 10 mM phosphate buffer pH 7.0, and then washed with 0-0.2
The enzyme was eluted using 10 mM phosphate buffer pH 7.0 containing M NaCl. The flow rate was 5 ml / min. The fraction having GDH activity was collected and 10 mM MO
It was dialyzed overnight against a PS-NaOH buffer (pH 7.0).
The electrophoretically uniform modified PQQGDH thus obtained
The protein was obtained. This was used as a purified enzyme preparation in the following Examples.

Example 4 Measurement of Enzyme Activity Enzyme activity was measured at room temperature with 10 mM MOPS.
Using PMS (phenazine methosulfate) -DCIP (2,6-dichlorophenolindophenol) in a NaOH buffer (pH 7.0), and tracking the change in the absorbance of DCIP at 600 nm using a spectrophotometer;
The rate of decrease in the absorbance was defined as the reaction rate of the enzyme. At this time, the enzyme activity for reducing 1 μmol of DCIP per minute was defined as 1 unit. The molar extinction coefficient of DCIP at pH 7.0 was 16.3 mM ⁻¹ .

Example 5 Evaluation of Selectivity for Glucose Substrate specificity of each crude purified enzyme preparation of modified PQQGDH was examined. The crude enzyme preparations of the wild type and each of the modified PQQGDH obtained in Example 3 were each 1 μMPQ
Q, holiation was performed for 1 hour or more in the presence of 1 mM CaCl ₂ . This was dispensed in 187 μl portions, and 3 μl of the active reagent (containing 6 mM DCIP, 600 mM PMS, 10 mM phosphate buffer pH 7.0) and the substrate were added. As a substrate, adjust the final concentration to 100 mM.
0 mM glucose, lactose and maltose in 1
0 μl was added, the mixture was incubated at room temperature for 30 minutes, and the enzyme activity was measured as in Example 4. The value was expressed as a relative activity with respect to the activity when glucose was used as a substrate, which was defined as 100. As shown in Table 2, all of the modified PQQGDHs of the present invention showed higher selectivity for glucose than lactose or maltose.

[0037]

[Table 2] Glucose lactose maltose wild type 100% 54% 58% Asp167Gly 100% 21% 0% Asp167His 100% 86% 0% Asp167Tyr 100% 57% 17% Asp167Ala 100% 68% 13% Asp167Glu 100% 32% 10% Asp167Lys 100% 54% 16% Asp167Asn 100% 62% 14% Asp167Gln 100% 59% 53% Asp167Val 100% 4% 36% Asp167Cys 100% 57% 22% Asp167Ser 100% 66% 15% Asp167Trp 100% 61% 15% Implementation Example 6 Evaluation of affinity of purified enzyme preparation for glucose Wild-type PQQGDH and Asp167Gl obtained in Example 3
u Using a crude enzyme preparation of modified PQQGDH, 1 μMPQQ, 1 mM CaCl ₂
Hollowed for over 1 hour in the presence. This was dispensed in 187 μl portions, and 3 μl of the active reagent (48 μl of 6 mM DCIP,
8 μl of 600 mM PMS, 10 mM phosphate buffer pH
7.0 16 μl) and 10 μl of each concentration of D-glucose solution were added, and the enzyme activity was measured at room temperature by the method described in Example 4. From a plot of substrate concentration versus enzyme activity,
Km and Vmax were determined. The Km value for glucose of wild-type PQQGDH was about 25 mM. In contrast, the Km value of Asp167Glu modified PQQGDH is about 55
mM. The activity of wild-type PQQGDH was 10
The specific activity of Asp167Glu at 0% was 90% or more.

Example 7 Glucose Assay Glucose was assayed using the modified PQQGDH. Asp167Glu modified PQQGDH, 1 μMPQQ,
In the presence of 1 mM CaCl _2, holation was performed for 1 hour or more, and glucose at various concentrations and 5 μMPQQ, 10 mM Ca
Cl Enzyme activity was measured ₂ presence. The method was based on the method for measuring the enzyme activity described in Example 4, and the DCIP was measured at 600 nm.
The change in absorbance was used as an index. As shown in FIG.
Using Asp167Glu modified PQQGDH, 1-500 m
Glucose could be quantified in the M range.

Example 8 Preparation and Evaluation of Enzyme Sensor 20 mg of carbon paste was added to 5 units of Asp167Glu modified PQQGDH, followed by freeze-drying. After this was mixed well, only the surface of the carbon paste electrode already filled with about 40 mg of carbon paste was filled and polished on filter paper. This electrode was treated in a 10 mM MOPS buffer (pH 7.0) containing 1% glutaraldehyde at room temperature for 30 minutes, and then treated with a 10 mM MOPS buffer containing 20 mM lysine.
Glutaraldehyde was blocked by treatment in MOPS buffer (pH 7.0) at room temperature for 20 minutes. The electrode was equilibrated in a 10 mM MOPS buffer (pH 7.0) at room temperature for 1 hour or more. The electrodes were stored at 4 ° C.

The glucose concentration was measured using the produced enzyme sensor. As shown in FIG. 4, glucose can be quantified in the range of 5 mM to 100 mM using the enzyme sensor on which the modified PQQGDH of the present invention is immobilized.

[0041]

The modified PQQGDH has high selectivity for glucose. Therefore, when an assay kit or an enzyme sensor is prepared using this enzyme, a higher selectivity is obtained as compared with the case where conventional natural PQQGDH is used. Obtainable.

[0042]

[Sequence Listing] Sequence Listing <110> Sode, Koji <120> Glucose Dehydrogenase <130> 001224 <160> 4 <210> 1 <211> 454 <212> PRT <213> Acinetobacter calcoaceticus <400> 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 340 345 350 350 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 LeuAsp Pro Thr Tyr Ser Thr Thr Tyr Asp Asp Ala Val Pro Met 385 390 395 400 400 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 <210> 2 <211> 1612 <212> DNA <213> Acinetobacter calcoaceticus < 400> 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 ttcaggtac a 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 g tattaagtt 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 <210> 3 <211> 20 <212> PRT <213> Acinetobacter calcoaceticus <220> <222> 4 <223> Xaa is any amino acid residue <400> 3 Ser Gln His Xaa Lys Ser Ser 1 5 <210> 4 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> primer for point mutation <400> 4 cctgactgat ggtgttttga tgaagg

[Brief description of the drawings]

FIG. 1 shows the plasmid p used in the present invention.
1 shows the structure of GB2.

FIG. 2 shows a method for preparing a mutant gene encoding the modified PQQGDH of the present invention.

FIG. 3 shows sv of the modified PQQGDH of the present invention.
Show the plot.

FIG. 4 shows an assay for glucose using the modified PQQGDH of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 9/04 C12Q 1/32 C12Q 1/00 1/54 1/32 G01N 33/66 C 1/54 ( C12N 1/21 G01N 33/66 C12R 1:01) // (C12N 1/21 (C12N 9/04 C12R 1:01) C12R 1:01) (C12N 9/04 C12N 15/00 ZNAA C12R 1:01) 5/00 A F-term (reference) 2G045 AA13 BB20 BB60 CA25 CA26 DA31 FA26 FA29 FB01 FB04 FB06 FB11 GC10 GC12 GC20 4B024 AA11 BA08 DA06 EA04 GA11 HA01 4B050 CC03 DD02 LL03 4B063 QA01 QA18 QQ02 QQ68 QR04 QR82 QS02 QS28 QS36 QS39 QX01 QX05 4B065 AA04Y AA26X AC14 BA02 CA28 CA46

Claims (13)

[Claims] 1. PQQGDH wherein the amino acid residue corresponding to the aspartic acid residue at position 167 of water-soluble PQQGDH derived from Acinetobacter calcoaceticus is substituted with another amino acid residue. 2. An amino acid sequence represented by SEQ ID NO: 1.
PQQGDH in which the aspartic acid residue at position 67 has been substituted with another amino acid residue. 3. The PQQGDH according to claim 1, wherein the other amino acid residue is glutamic acid. 4. A PQQGDH comprising the sequence: Ser Gln His Xaa Lys Ser Ser, wherein Xaa is any natural amino acid residue other than Asp. 5. The PQQ of claim 3, wherein Xaa is Glu.
GDH. 6. The PQQGDH according to claim 1, which has a higher selectivity for glucose compared to wild-type PQQGDH. 7. The PQQ according to claim 1, wherein:
Gene encoding GDH. 8. A vector containing the gene according to claim 7. 9. A transformant containing the gene according to claim 7. 10. The transformant according to claim 12, wherein the gene according to claim 7 is integrated into a main chromosome. 11. A method for producing water-soluble PQQGDH, comprising culturing the transformant according to claim 9, and preparing a water-soluble fraction from the cells. 12. The PQ according to claim 1,
A glucose assay kit containing QGDH. 13. The PQ according to claim 1,
Glucose sensor containing QGDH.