WO2006095758A1

WO2006095758A1 - Method of avoiding substrate inhibition by pqqgdh

Info

Publication number: WO2006095758A1
Application number: PCT/JP2006/304440
Authority: WO
Inventors: Tadanobu Matsumura
Original assignee: Toyo Boseki Kabushiki Kaisha
Priority date: 2005-03-11
Filing date: 2006-03-08
Publication date: 2006-09-14
Also published as: JP2006280360A

Abstract

[PROBLEMS] To provide a method of avoiding any substrate inhibition by pyrroloquinoline quinone dependent glucose dehydrogenase. [MEANS FOR SOLVING PROBLEMS] With respect to the measurement of glucose in a system containing pyrroloquinoline quinone dependent glucose dehydrogenase, there is provided a method of reducing any substrate inhibition during glucose measurement, characterized in that the pH value at measuring reaction falls within the acid region.

Description

Specification

How to avoid substrate inhibition of PQQGDH

Technical field

[0001] The present invention relates to a method for avoiding substrate inhibition of pyroguchi quinoline quinone-dependent glucose dehydrogenase.

Background art

[0002] Pyroguchi quinoline quinone-dependent gnolecose dehydrogenase (also referred to as PQQGDH in this application) is gnorecose dehydrogenase (GDH) using pyroguchi quinoline quinone (PQQ) as a coenzyme. Since it catalyzes the reaction that oxidizes gnolecose to produce dalconolatatone, it can be used for blood glucose measurement. Blood glucose concentration is an extremely important index for clinical diagnosis as an important marker of diabetes. Currently, biosensors are the mainstream for measuring blood glucose levels S, and pyroguchi quinoline quinone-dependent glucose dehydrogenase is attracting attention as an enzyme used to measure blood glucose levels. . As such PQQGDH, for example, it was found that Acinetobacter baumannii NCIMB11517 strain S, pyroguchi quinoline quinone-dependent glucose dehydrogenase was produced, and a gene cloning and high expression system was constructed (for example, , See Patent Document 1).

Patent Document 1: Japanese Patent Laid-Open No. 11-243949

Brief Description of Drawings

[0003] [Fig.1] Relationship between glucose concentration and PQQGDH reaction rate in PIPES buffer, ρΗ6.5

[Figure 2] Relationship between glucose concentration and PQQGDH reaction rate in PIPES buffer, ρΗ5 · 5

[Figure 3] Relationship between glucose concentration and PQQGDH reaction rate in 2-ketoglutarate buffer, ρΗ6.5

[Figure 4] Relationship between glucose concentration and PQQGDH reaction rate in 2-ketoglutarate buffer, ρΗ5 · 5 [Figure 5] Relationship between gnolecose concentration and PQQGDH reaction rate in malate buffer, ρΗ6 · 5

[Figure 6] Relationship between gnolecose concentration and PQQGDH reaction rate in malate buffer, ρΗ5 · 5

[Fig.7] Relationship between glucose concentration and PQQGDH reaction rate in succinate buffer, ρΗ6.5

[Fig.8] Relationship between glucose concentration and PQQGDH reaction rate in succinate buffer, ρΗ5.5

[Figure 9] Relationship between glucose concentration and PQQGDH reaction rate in dartrate buffer, ρΗ6.5

[Figure 10] Relationship between glucose concentration and PQQGDH reaction rate in dartrate buffer, ρΗ5.5

[Fig. 11] Relationship between glucose concentration and PQQGDH reaction rate in MES buffer, ρΗ6 · 5

[Figure 12] Relationship between glucose concentration and PQQGDH reaction rate in MES buffer, ρΗ5 · 5

[Figure 13] Relationship between glucose concentration and PQQGDH reaction rate in MOPSO buffer, ρΗ7.0

[Fig.14] Relationship between glucose concentration and PQQGDH reaction rate in MOPSO buffer, ρΗ6 · 5

[Figure 15] Relationship between glucose concentration and PQQGDH reaction rate in MOPS buffer, ρΗ7.5

[Figure 16] Relationship between glucose concentration and PQQGDH reaction rate in MOPS buffer, pH 6.5

[Fig.17] Relationship between glucose concentration and PQQGDH reaction rate in fumarate buffer, pH 6.5

[Figure 18] Relationship between glucose concentration and PQQGDH reaction rate in fumarate buffer, pH 5.5 [Fig.19] Concentration of each component in the reaction solution in the gnolecose measurement system

Disclosure of the invention

Problems to be solved by the invention

[0004] Pyromouth quinoline quinone-dependent glucose dehydrogenase catalyzes the reaction that oxidizes D-glucose to produce D-darcono 1,5-latataton, and is not affected by dissolved oxygen in the reaction system. Because it has enzyme properties that do not require the addition of coenzymes, it is expected to have a wide range of uses such as blood glucose biochemical diagnostic agents as well as blood glucose sensors.

Means for solving the problem

[0005] In order to solve the above-mentioned problems, the present inventors have intensively studied the cause, and as a result, the reactivity to ferricyanide used as an electron mediator in a blood glucose sensor is generally low. I understood it.

We further investigated this point and clarified that the reason for the low reactivity to ferricyanide ions was that the buffer conditions were affected by substrate inhibition near neutrality.

To date, there has been a report on a method for avoiding substrate inhibition of PQQGDH, and Patent Document 2 has reported studies using PQQGDH modification means at the gene level, but the mechanism of substrate inhibition has also been disclosed. There was no suggestion, and it was not even mentioned as a means to resolve substrate inhibition from the viewpoint of measurement reaction conditions.

Patent Document 2: W003Zl06668

[0006] As a result of further diligent research, the present inventors have made a search for a simpler avoidance method for substrate inhibition from a viewpoint different from the past measures, and as a result, made the pH of the reaction conditions for the measurement of gnolecose acidic. As a result, it was clarified that the substrate inhibition of PQQGDH can be avoided, and the present invention has finally been completed. That is, the present invention

Item 1. A method for reducing substrate inhibition in dalcose measurement, characterized in that the pH during measurement reaction is acidic in a method for measuring gno-record in a system containing pyroguchi quinoline quinone-dependent glucose dehydrogenase Item 2. The glucose measurement method according to Item 1, comprising ferricyanide ion as a mediator

Item 3. Glucose measuring reagent with reduced substrate inhibition characterized by containing pyroguchi quinoline quinone-dependent glucose dehydrogenase and acidic ρΗ during the measurement reaction Item 4. Ferricyanide ion as a mediator The glucose measurement reagent according to Item 3, comprising

Item 5. Glucose assay kit with reduced substrate inhibition characterized by containing pyroguchi quinoline quinone-dependent glucose dehydrogenase and ρΗ at the time of measurement reaction being acidic Item 6. Ferricyanide as a mediator The glucose assembly kit according to Item 5, comprising an ion

Item 7. Glucose sensor with reduced substrate inhibition characterized by containing pyroguchi quinoline quinone-dependent glucose dehydrogenase and acidic ρΗ during the measurement reaction Item 8. Felicyanide ion as a mediator The glucose sensor according to Item 7, comprising

It is.

The invention's effect

[0007] Avoidance of substrate inhibition according to the present invention enables high-concentration glucose measurement using a gnolecose measuring reagent, a gnolecose assembly kit, and a gnolecose sensor.

BEST MODE FOR CARRYING OUT THE INVENTION

[0008] Hereinafter, the present invention will be described in detail.

[0009] PQQGDH, which can be applied to the method of the present invention, coordinates Pyroguchi quinoline quinone as a coenzyme and oxidizes D-glucose to produce D-darcono 1,5-latataton. It is an enzyme that catalyzes (ECl.

[0010] PQQGDH that can be applied to the method of the present invention is derived from, for example, Acinetobacter calcoaceticus LMD79.41 (A. M. Cleton- Jansen et al., J. Bacteriol., 170, 2121 (1988) and Mol. Gen. Gen et., 217, 430 (1989)), derived from Escherichia coli (A. M Cleton—Jansen et al., J. Bacteriol., 172, 6308 (1990)), derived from Gluconobacter oxydans (Mol. Gen. Genet., 229, 20 6 (1991) And those derived from Acine tobacter baumanni NCIMB11517 reported in Patent Document 1.

However, it is difficult to modify the membrane enzyme present in Escherichia coli to make it soluble, and the origin is to select soluble PQQGDH such as Acinetopacter 'Calcoaceticus or Acinetopacter baumannii preferable.

In addition, Acinetobacter baumannii (Acinetobacter baumannii) NCIMB 1151 7fe¾ 冃 11, Acinetobacter calcoaceticus.

[0011] PQQGDH that can be applied to the method of the present invention is not limited to those exemplified above, as long as it has glucose dehydrogenase activity. Other amino acid residues may be added.

Such modifications can be easily carried out by those skilled in the art using known techniques in the art. For example, it encodes the protein to introduce site-specific mutations into the protein.

Various methods for substituting the base sequence of the gene to be described are described by Sambrook et al., Molecular Cloning; A Laboratory Manual, Part 2 (1989) Cold Spring Harbor Laboratory Press, New York.

[0012] For these PQQGDH, for example, commercially available products such as Toyobo GLD-321 can be used. Alternatively, it can be easily produced by those skilled in the art using known techniques in the technical field.

[0013] For example, the above-mentioned natural microorganisms that produce PQQGDH, or the gene encoding natural PQQGDH as it is or after mutation, expression vectors (many are known in the art) For example, a plasmid is inserted into a suitable host (many are known in the art, such as E. coli), and the transformant is cultured and centrifuged from the culture. After harvesting the cells, etc., destroy the cells by mechanical methods or enzymatic methods such as lysozyme, and if necessary, solubilize by adding a chelating agent such as EDT A or a surfactant. Water soluble, including PQQGDH A fraction can be obtained. Alternatively, the expressed PQQGDH can be secreted directly into the culture medium by using an appropriate host vector system.

[0014] The PQQGDH-containing solution obtained as described above is subjected to, for example, vacuum concentration, membrane concentration, salting-out treatment such as ammonium sulfate or sodium sulfate, or hydrophilic organic solvents such as methanol, ethanol, acetone and the like. Precipitate by the fractional precipitation method. Heat treatment and isoelectric point treatment are also effective purification means. In addition, purified PQQGDH can be obtained by performing gel filtration using an adsorbent or gel filter, adsorption chromatography, ion exchange chromatography, and affinity chromatography. The purified enzyme preparation is preferably purified to such an extent that it shows a single band on electrophoresis (SDS-PAGE).

[0015] Before and after the above step, in order to improve the ratio of holo-type PQQGDH to the total GDH enzyme protein, a heat treatment of preferably 25 to 50 ° C, more preferably 30 to 45 ° C may be performed. .

[0016] The concentration of PQQGDH in the present invention is not particularly limited.

[0017] The enzyme activity of PQQGDH can be measured by the following method.

Method for measuring PQQGDH enzyme activity

(1) Measurement principle

D—Glucose + PMS + PQQGDH → D—Dalcono 1, 5—Lataton + PMS ed)

2PMS (red) + NTB → 2PMS + diformazan

The presence of diformazan formed by the reduction of nitrotetrazolium blue (NTB) with phenazine methosulfate (PMS) (red) was measured spectrophotometrically at 570 nm.

(2) Unit definition

One unit refers to the amount of PQQGDH enzyme that forms 0.5 mmol of diformazan per minute under the conditions described below.

(3) Method

reagent A. D—glucose solution: 0.5M (0.9 g D—glucose (molecular weight 180 · 16) / 10ml H20)

B. PIPES—NaOH buffer, pH 6.5: 50 mM (1.51 g of PIPES (molecular weight 302.36) suspended in 60 mL of water, dissolved in 5 N NaOH, 2. 2 ml of 10% Triton X_100 (Use 5N NaOH for 25. Adjust the ptl to 6.5 ± 0.05 with C and add 100 ml with water)

C. PMS solution: 3.0mM (9.19mg phenazine methosanolate (molecular weight 817.65) / l0mlH2O)

D. NTB solution: 6.6 mM (53. 96 mg of double-mouthed terazolium bunoleol (molecular weight 817.65) / l0mlH2O)

E. Enzyme Diluent: ImM CaC12, 0.1% Triton X-100, 0.1 mM BSA-containing 50 mM PIPES _Na 0H buffer (pH 6.5)

Steps

Prepare the following reaction mixture in a light-proof bottle and store on ice (prepared at the time of use)

1. 8ml D-glucose solution (A)

24. 6ml PIPES—NaOH buffer (pH 6.5) (B)

2. 0ml PMS solution (C)

1. 0ml NTB solution (D) Concentrations in the above assembly are as follows.

PIPES buffer 42 mM

D—Glucose 30 mM

PMS 0.20mM

NTB 0.22mM

3. Put 0ml reaction mixture into a test tube (plastic) and preheat at 37 ° C for 5 minutes

0.1 ml of enzyme solution was added and gently inverted to mix. The increase in absorbance for water at 570 nm was recorded for 4-5 minutes with a spectrophotometer while maintaining at 37 ° C, and the A OD per minute from the initial linear portion of the curve was calculated (OD test). At the same time, a blank (ΔOD blank) was measured by repeating the same method except that an enzyme diluent (E) was added instead of the enzyme solution.

The enzyme powder was dissolved in ice-cold enzyme diluent (E) immediately before the assembly and diluted to 0.1 -0.8 U / ml with the same buffer (use a plastic tube for the adhesion of the enzyme). Is preferred).

Tojo

Activity is calculated using the following formula:

UZml = {A OD / min (A OD test 1 00 blank) (1 ^ 7 (20. 1 X 1. O XVs)

U / mg = (U / ml) X l / C

Vt: Total volume (3. lml)

Vs: Sampnore volume (1.0ml)

20. 1: 1/2 mmol molecular extinction coefficient of diformazan

1. 0: Optical path length (cm)

df: dilution factor

C: Enzyme concentration in solution (c mg / ml)

[0018] In the method of the present invention, the pH at the time of measurement is acidic. The pH is less than 7.0 and is not particularly limited, but the upper limit is preferably 6.5 or less, more preferably 6.0 or less, and even more preferably 5.5 or less in the present invention. In the present invention, the lower limit is preferably pH 3.0 or higher, more preferably pH 3.5 or higher. Furthermore, the preferred range in the present invention is pH 3.5 to 5.5.

[0019] In the method of the present invention, it is possible to use various buffers to make the pH during measurement acidic. Such a buffer is not particularly limited as long as it has a buffer capacity capable of keeping the pH acidic. Commonly used buffers include tris hydrochloric acid, boric acid, phosphoric acid, acetic acid, succinic acid, succinic acid, phthalic acid, maleic acid, glycine and their salts, MES, Bis_Tris, ADA, PIPES , ACES, MOPS 0, DES buffer such as BES, MOPS, TES, HEPES, etc. Do not form insoluble salts with calcium, buffer is preferred.

Only one of these may be applied, or two or more may be used. Further, one or more composite compositions including those other than the above may be used.

The concentration of these additives is not particularly limited as long as it has a buffering capacity, but the preferable upper limit is 10 mM or less, more preferably 50 mM or less. A preferred lower limit is 5 mM or more.

The content of the buffer in the lyophilized product is not particularly limited, but is preferably 0.1% (weight ratio) or more, particularly preferably 0.:! To 30% (weight ratio). Used in the range of.

These can use various commercially available reagents.

[0020] These buffers may be added at the time of measurement, and may be preliminarily contained when preparing a reagent for measuring gnolecose, a gnolecose assembly kit or a glucose sensor described later. . In this case, the liquid state, the dry state, etc. are not limited, and it is sufficient to function during measurement.

[0021] Substrate inhibition as used in the present invention refers to a phenomenon in which the reaction rate decreases at a constant concentration when the glucose (substrate) concentration of the sample to be measured is increased. When samples with various darose concentrations are measured, the glucose concentration immediately before the reaction rate decreases is measured.

It is regarded as “the highest concentration at which substrate inhibition does not occur”.

[0022] In addition, "reducing substrate inhibition" means increasing the "maximum concentration at which substrate inhibition does not occur". In practice, however, normoglycemia is about 5 mM (90 mg / dl), and hyperglycemia is present. Value is 10mM

Judging from the fact that it is about (180mgZdl), it is 30mM (54

If no substrate inhibition is observed up to (OmgZdl) (that is, if the “maximum concentration at which substrate inhibition does not occur” is 30 mM or more), it is practically sufficient.

Note that the measurement range of glucose is completely different from the “upper limit of the maximum concentration at which substrate inhibition does not occur”.

[0023] The effect of the present invention becomes more remarkable in a system including a mediator. The mediator that can be applied to the method of the present invention is not particularly limited. (PMS) and 2,6-dichlorophenol indophenol (DCPIP), PMS and nitroblue tetrazolium (NBT), DCPIP alone, ferricyanide ion (as a compound) Potassium ferricyanide, etc.) alone, and Huekousen alone. Of these, ferricyanide ions (such as ferricyanium potassium as the compound) are preferred.

Since there are various differences in sensitivity among these mediators, it is not necessary to uniformly define the concentration of addition, but in general, an additive of ImM or higher is desirable.

[0024] These mediators may be added at the time of measurement, or may be included in advance when a glucose measurement reagent, a glucose assay kit, or a glucose sensor described later is prepared. In that case, the liquid state, the dry state, etc. are not asked and the measurement is not performed.

It should be dissociated at the time of the reaction so as to be in an ionic state.

[0025] In the present invention, various components can coexist as necessary. For example, surfactants, stabilizers, excipients and the like may be added.

[0026] For example, PQQGDH can be further stabilized by adding calcium ions or salts thereof, amino acids such as glutamic acid, glutamine, and lysine, and serum albumin.

[0027] For example, PQQGDH can be stabilized by containing calcium ions or calcium salts. Examples of the calcium salt include calcium chloride, calcium acetate, calcium salt of inorganic acid such as calcium citrate, and calcium salt of organic acid. In the aqueous composition, the calcium ion content is preferably 1 X 10-4 to 1 X 10-2M.

[0028] The stabilizing effect of containing calcium ions or calcium salts is further improved by containing an amino acid selected from the group consisting of gnoretamic acid, glutamine and lysine. The amino acid selected from the group consisting of glutamic acid, glutamine and lysine may be one type or two or more types. Add bovine serum albumin (BSA) and ovalbumin (OVA).

[0029] Alternatively, (1) aspartic acid, gnoretamic acid, a-ketoglutaric acid, malic acid, a-ke PQQGDH can be stabilized by the coexistence of one or more compounds selected from the group consisting of togluconic acid, _a- cyclodextrin and their salts, and (2) albumin.

[0030] In the present invention, glucose can be measured by the following various methods.

The reagent for measuring glucose, the glucose assay kit, and the glucose sensor of the present invention can take various forms such as liquid (aqueous solution, suspension, etc.), powdered by vacuum drying or spray drying, freeze drying, etc. . The freeze-drying method is not particularly limited, but should be performed according to conventional methods. The composition containing the enzyme of the present invention is not limited to a lyophilized product, but may be a solution in which the lyophilized product is redissolved.

[0031] Glucose measuring reagent

The reagent for measuring glucose of the present invention typically includes a reagent necessary for measurement such as PQQGDH, a buffer solution, a mediator, a glucose standard solution for preparing a calibration curve, and a usage guideline. The kit of the present invention can be provided, for example, as a lyophilized reagent or as a solution in a suitable storage solution. Preferably, the PQQGDH of the present invention can be provided in the form of a force apoenzyme provided in a holified form and can be holoed into a used B temple.

[0032] Glucose assembly kit

The glucose assay kit of the present invention typically includes reagents necessary for measurement such as PQQGDH, buffer, and mediator, a glucose standard solution for preparing a calibration curve, and usage guidelines. The kit of the present invention can be provided, for example, as a lyophilized reagent or as a solution in a suitable storage solution. Preferably, the PQQGDH of the present invention is provided in the form of a holo, but it can also be provided in the form of an apoenzyme and holoed at the time of use.

[0033] Glucose sensor

In the glucose sensor of the present invention, a carbon electrode, a gold electrode, a platinum electrode, or the like is used as an electrode, and PQQGLD is immobilized on this electrode. Examples of the immobilization method include a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a method of using a photocrosslinking polymer, a conductive polymer, a redox polymer, or the like. It can be fixed in a polymer or adsorbed and fixed on an electrode together with an electron mediator represented by Huekousen or its derivatives, or a combination thereof.

. Preferably, the PQQGDH of the present invention may be immobilized in the form of a force apoenzyme that is immobilized on the electrode in a holo form, and PQQ may be supplied as a separate layer or in solution. Typically, PQQGDH of the present invention is immobilized on a carbon electrode using dartalaldehyde, and then treated with a reagent having an amine group to block the gnoretaraldehyde.

[0034] Measurement of the gnolecose concentration can be performed as follows. Put the buffer in the thermostatic cell, and keep CaC12 and mediator at a constant temperature. As the mediator, potassium ferricyanide, phenazine methosulfate, or the like can be used. As the working electrode, an electrode on which the PQQGDH of the present invention is immobilized 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 current is measured. The glucose concentration in the sample can be calculated according to the calibration curve prepared with the standard concentration of gnolecose solution.

Example

Hereinafter, the present invention will be described in more detail based on examples. However, the present invention is not limited to the examples.

[0036] Example 1

Confirmation of substrate inhibition using the gnolecose measurement system

Measurement principle

PQQGDH

D-glucose + ferricyanide ion →

D-Dalcono 1,5-Lataton + ferrocyanide ion The presence of ferrocyanide ion produced by reduction of ferricyanide ion was confirmed by measuring the decrease in absorbance at a wavelength of 420 nm by spectrophotometry.

(2) Method

reagent A. PIPES—NaOH buffer, ρΗ6 · 5: 50 mM (1.51 g PIPES (molecular weight 302. 36) suspended in 60 mL of water was dissolved in 5 N NaOH, and 2 ml of 10% Triton X— 1 Calorie 00. Adjust the pH to 6 · 5 ± 0 · 05 at 25 ° C using 5N NaOH, and add water to make 100 ml.)

B. Potassium ferricyanide solution: 50 mM (0.165 g potassium ferricyanide (molecular weight 32

9. 25) was dissolved in 10ml distilled water)

C. PQQGDH solution: 8000 U / ml (about lOOmg PQQGDH (Toyobo Co., Ltd .: GLD-321) dissolved in 10 ml of distilled water) Sample

D-Gnolecose solution: Based on 1500mM glucose solution prepared at 150, 300, 450, 600, 750, 900, 1050, 1200, 1350 and 1500mM respectively 1/10, 2/10, 3/10, 4 /

(10, 5/10, 6/10, 7/10, 8/10, 9/10)

1. Prepare the following reaction mixture in a light-proof bottle and store it on ice (preparation before use)

49. 6ml PIPES—NaOH buffer (pH 6.5) (A)

4. 0ml potassium ferricyanide solution (B)

5. 6ml (C)

2. 3. 0 ml of the reaction mixture was placed in a test tube (plastic) and pre-warmed at 37 ° C for 5 minutes.

3. Add 0.1 ml glucose solution and mix gently.

4. Record the decrease in absorbance for water at 420nm at 37 ° C for 1-3 minutes with a spectrophotometer and calculate the A OD per minute from the initial linear part of the curve (A OD test)

At the same time, the same method was performed except that distilled water was added instead of the glucose solution, and a blank (ΔOD blank) was measured. The above operation was performed using a glucose solution having a concentration of 150 mM to 1500 mM.

[0037] FIG. 19 shows the concentration of each component in the reaction solution.

[0038] Calculation

The change in absorbance per unit time was determined by calculating A ODZmin (A0D test 1 ΔOD blank).

The horizontal axis of the graph plots the glucose concentration in the reaction solution, and the vertical axis plots Δ OD / min corresponding to each glucose concentration.

As shown in Fig. 1, under this condition, AODZmin with a glucose concentration of 10 mM as the upper limit, that is, the reaction rate does not increase, and the reaction rate decreases with increasing glucose concentration, so the effect of substrate inhibition can be confirmed. .

[0039] Example 2

Confirmation of substrate inhibition avoidance by changing the pH condition of the gnolecose measurement system

Based on the procedure of Example 1, the pH of the buffer was changed from 6.5 to 5.5. All other conditions were in accordance with Example 1.

As shown in Fig. 2, it was found that the reaction rate, which had reached the peak at 1 OmM, continued to increase to a high glucose concentration and increased to 40 mM by shifting the buffer pH from near neutral to acidic. It was. After that, the same speed was maintained and the reaction speed was not decreased. In addition, an overall increase in the reaction rate was also observed.

Thus, it became clear that substrate inhibition can be avoided by shifting the pH of the buffer from near neutral to acidic.

[0040] Example 3

Based on the methods of Examples 1 and 2, when 2_ketognoletaric acid, malic acid, succinic acid, gnoretalic acid, and fumaric acid are used as one kind of buffer, and when DES buffers MES, MOPS 0, and MOPS are used Confirmation was carried out. Regarding Dat buffer, MOPSO was confirmed at pH 7.0 and pH 6.5, and MOPS was confirmed at pH 7.5 and pH 6.5, referring to the pH buffer range of each.

As shown in Fig. 3 to Fig. 18, the pH shifts to the acidic side in other buffer types as well. It was confirmed that the substrate inhibition could be reduced. Thus, it can be said that this effect is generally recognized without being limited to a specific buffer type. Industrial applicability

The avoidance of substrate inhibition according to the present invention enables high-concentration glucose measurement with a gnolecose measurement reagent, a glucose assembly kit and a gnolecose sensor.

Claims

The scope of the claims

[1] A method for reducing substrate inhibition in glucose measurement, wherein glucose is measured in a system containing pyroguchi quinoline quinone-dependent gnolecose dehydrogenase, wherein the pH during the measurement reaction is acidic.

[2] The method for measuring gnolecose according to claim 1, comprising ferricyanide ions as mediators.

[3] A glucose measurement reagent with reduced substrate inhibition, characterized by comprising pyroguchi quinoline quinone-dependent gnolecose dehydrogenase and having an acidic pH during the measurement reaction.

[4] The gnolecose measurement according to claim 3, comprising ferricyanide ions as mediators.

[5] A gnolecose assay kit with reduced substrate inhibition, characterized by containing pyroguchi quinoline quinone-dependent gnolecose dehydrogenase and having an acidic pH during the measurement reaction.

6. The glucose assay kit according to claim 5, comprising ferricyanide ion as a mediator.

[7] A gnolecose sensor with reduced substrate inhibition, characterized by containing a pyroguchi quinoline quinone-dependent gnolecose dehydrogenase and having an acidic pH during the measurement reaction.

[8] The gnolecose sensor according to claim 7, comprising ferricyanide ions as mediators.