WO2006046524A1

WO2006046524A1 - Pqq-dependent glucose dehydrogenase composition

Info

Publication number: WO2006046524A1
Application number: PCT/JP2005/019532
Authority: WO
Inventors: Tadanobu Matsumura; Kayoko Kajitani
Original assignee: Toyo Boseki Kabushiki Kaisha
Priority date: 2004-10-27
Filing date: 2005-10-25
Publication date: 2006-05-04
Also published as: TW200630492A

Abstract

[PROBLEMS] To provide a practically useful glucose measuring system whereby the stability of an enzyme can be ensured in measuring glucose with the use of PPQ-dependent glucose dehydrogenase. [MEANS FOR SOLVING PROBLEMS] A pyrroloquinoline quinone-dependent glucose dehydrogenase composition characterized by being free from protein component other than protein components originating in the host; or a pyrroloquinoline quinone-dependent glucose dehydrogenase composition which exclusively contains calcium or a calcium salt and a buffering agent in addition to protein components originating in the host.

Description

Specification

PQQ-dependent glucose dehydrogenase composition

Technical field

[0001] The present invention relates to a stabilized pyroguchi quinoline quinone-dependent glucose dehydrogenase composition. (Hereinafter, pyroguchi quinoline quinone is also referred to as PQQ, gnolecose dehydrogenase as GDH, and pyroguchi quinoline quinone-dependent glucose dehydrogenase as PQQGDH.)

Background art

[0002] As a method for measuring glucose in specimens such as serum and urine, a measurement system using PQQ-dependent gnolecose dehydrogenase that requires PQQ as a coenzyme is known (for example, Non-Patent Document 1). Although this measurement system can be applied to reactions in a solution, it is generally in the form of a gnolecose sensor that is widely used (for example, Patent Document 1). In such a glucose sensor, components necessary for the reaction such as PQQ-dependent glucose dehydrogenase are often immobilized on the sensor in a dry state.

Non-Patent Document 1: Methods in Enzymology, Vol. 89 (1982) p20 (Academic

Press, Inc)

Patent Document 1: Patent Publication 2003—207475

Disclosure of the invention

Problems to be solved by the invention

[0003] Generally, enzyme preparations are added with some kind of stabilizer for the purpose of improving or maintaining their stability, and the same applies to PQQ-dependent glucose dehydrogenase preparations. However, in the case of PQQ-dependent gnolecose dehydrogenase, if the enzyme is applied to the sensor, the stabilizer may affect the measured value and cause unforeseen problems such as loss of accuracy. . In consideration of the solubility of the components required for the reaction at the time of measurement, it is desirable that the enzyme preparation having the same activity be a small amount. For these reasons, it is desirable that the added stabilizer is as small as possible, and it is desirable to ensure the stability and accuracy of the enzyme preparation as well as the sensor. An object of the present invention is to achieve both the stability of an enzyme preparation and the reduction of factors that impair the accuracy of measurement when applied to a sensor.

Means for solving the problem

[0004] The present inventor has conducted various studies on a stabilizer for PQQ-dependent glucose dehydrogenase. As a result, the inventors have found a composition that can ensure stability without using a biological substance, and have reached the present invention. .

[0005] The outline of the present invention is as follows. That is,

[Section 1

A pyroguchi quinoline quinone-dependent gnolecose dehydrogenase composition comprising no protein component other than a host-derived protein component.

[Section 2]

The pyroguchi quinoline quinone-dependent glucose dehydrogenase composition according to claim 1, which contains only calcium or a calcium salt and a buffer in addition to the protein component derived from the host.

[Section 3

The pyroguchi quinoline quinone-dependent glucose dehydrogenase composition according to claim 1, which contains only calcium or calcium salt, pyroguchi quinoline quinone, and a buffer in addition to the protein component derived from the host.

[Section 4

A method for stabilizing pyroguchi quinoline quinone-dependent gnolecose dehydrogenase in a pyroguchi quinoline quinone-dependent gnolecose dehydrogenase composition, which does not contain a protein component other than a host-derived protein component.

[Section 5]

5. The pyroguchi quinoline quinone-dependent gnolecose dehydrogenase composition according to claim 4, characterized by containing only calcium or a calcium salt and a buffer in addition to the protein component derived from the host. A method for stabilizing a dependent gnolecose dehydrogenase.

[Section 6] A method for producing a stabilized pyroguchi quinoline quinone-dependent gnolecose dehydrogenase composition, characterized by not containing a protein component other than a host-derived protein component.

[Section 7]

7. The method for producing a stabilized pyroguchi quinoline quinone-dependent glucose dehydrogenase composition according to claim 6, wherein only the calcium or calcium salt and the buffer are contained in addition to the protein component derived from the host. .

[Section 8

A glucose assembly comprising the glucose dehydrogenase composition according to claim 1.

[Item 9

A glucose sensor comprising the glucose dehydrogenase composition according to claim 1. The invention's effect

[0006] According to the present invention, it is possible to provide a Dalcors measurement system that ensures the stability of the enzyme and is highly practical in measuring glucose by a sensor using a PQQ-dependent glucose dehydrogenase. In addition, the stability of the enzyme and the accuracy of the measurement can be compatible, and a genolecose measuring system excellent in practicality can be provided.

Brief Description of Drawings

[0007] [FIG. 1] Comparison of glucose dehydrogenase residual activity after storage in each composition of Example 2

[Fig. 2] Comparison of glucose dehydrogenase residual activity after storage in each composition of Example 3.

BEST MODE FOR CARRYING OUT THE INVENTION

[0008] In the present invention, PQQ-dependent glucose dehydrogenase is an enzyme that coordinates PQQ as a coenzyme, and oxidizes D-glucose to produce D-darcono 1,5-latataton. It is an enzyme that catalyzes (EC1. 1. 5. 2 (formerly EC1. 1. 99. 17)), and there are no particular restrictions on its origin or structure.

For example, in microorganisms, the genus Acinetobacter, the genus Pseudomonas, the Burkholde Produced from ria, Gluconobactor, or Acetobactor.

For example, Acinetobacter baumannii (for example, see Patent Document 1), Acinetobacter calcoaceticus (for example, see Non-Patent Documents 1 and 2), Syudomonas. Aeruginosa (Ps eudomonasaeruginosa), Gerhard ¹ Tomonasu 'putida (Pseudomonas putida Li, Gerhard Domonasu' unloading Leo column sense (Pseudomonas fluorescens), Gunorekonono Kuta one 'O Kishidansu (Gluconobacter oxydans) (e.g., see non-Patent Document 3.) Oxidatives such as oyster fungus, Agrobacterium radiobacter, Escherichia coli (see, for example, non-patent document 4), Klebsiella aerogenes, etc. Enterobacteriaceae can Burkholderia 'Sepashia (B urkhorderia cepacia the kick force ^s like Li.

For example, it is produced from Acinetobacter baumannii NCIMB11517 strain, Acinetobacter calcoaceticus IF01552 strain, Acinetobacter calcoaceticus LMD79.41 strain, etc. (Patent Documents 2, 3, and 4).

Non-patent document 1: Α · M. Cleton—Jansen et al., J. Bacteriol., 170, 2121 (1988) Non-patent document 2: Μο1 · Gen. Genet., 217, 430 (1989)

Non-Patent Document 3: Μο1Gen. Genet., 229, 206 (1991)

Non-Patent Document 4: M. Cleton—Jansen et al., J. Bacteriol., 172, 6308 (1990) Patent Document 2: JP-A-11 243949

Patent Document 3: JP 2004-173538 A

Patent Document 4: Special Table 2004— 512047

PQQGDH that can be applied to the present invention may have a part of other amino acid residues deleted or replaced with those exemplified above, as long as it has gnoreucose dehydrogenase activity. Also, 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, various methods for substituting or inserting the base sequence of a gene encoding a protein in order to introduce site-specific mutation into the protein include S Ambrook et al., Molecular Cloning; A Laboratory Manual, Part 2 and (1989) Cold Spring Harbor Laboratory Press, New York.

[0010] These PQQGDHs can be easily produced by those skilled in the art using known techniques in the art.

For example, an expression vector (many of which are known in the art, after the above-mentioned natural microorganism producing PQQGDH or the gene encoding natural PQQGDH is directly or mutated) And then transforming the transformant transformed into an appropriate host (many are known in the art, eg, E. coli), and centrifuging it from the culture. After the body is recovered, the cells are destroyed by a mechanical method or an enzymatic method such as lysozyme, and if necessary, a chelating agent such as EDTA or a surfactant is added to solubilize the PQQGDH. A water-soluble fraction can be obtained. Alternatively, the expressed PQQGDH can be secreted directly into the culture medium by using an appropriate host vector system.

The PQQGDH-containing solution obtained as described above is subjected to, for example, vacuum concentration, membrane concentration, salting out treatment such as ammonium sulfate and sodium sulfate, or fractional precipitation using a hydrophilic organic solvent such as methanol, ethanol, acetone, etc. It should be precipitated by the law. 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).

Before and after the above steps, heat treatment at 25 to 50 ° C, more preferably 30 to 45 ° C may be performed to improve the ratio of holo-type PQQGDH to the total GDH enzyme protein.

[0011] The PQQGDH produced as described above contains substantially no protein component other than the protein component derived from the host.

Alternatively, for example, a commercially available enzyme such as GLD-321 manufactured by Toyobo Co. It is possible to manufacture S.

[0012] The composition of the present invention further undergoes a process other than "recovering host-derived protein" to PQQGDH containing no protein component other than the host-derived protein component produced as described above. Can be obtained. For example, it is not necessary to add anything to PQQGDH, and it can be achieved by adding various components other than host-derived protein components.

The form of the composition can take various forms such as an aqueous composition such as liquid (aqueous solution, suspension, etc.), powdered by vacuum drying or spray drying, freeze drying, etc., but is not particularly limited. . The lyophilization method is not particularly limited and may be performed according to a conventional method. 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.

[0013] Alternatively, the composition of the present invention basically comprises the enzyme, calcium or a calcium salt, and a buffer. Further, after containing these, pyro-quinoline quinone, amino acid, saccharide or organic acid may be further added. In addition, the enzyme composition of the present invention may be an aqueous composition or a lyophilized product as long as it contains these.

Examples of the calcium supply form include calcium salts of inorganic acids or organic acids such as calcium chloride, calcium acetate, calcium citrate and the like. Of these, calcium chloride is preferred. In the powder composition, the calcium content (W / W) is desirably 0.05% to 5%, and more preferably 0.1% to 0.5%.

As the buffering agent, those generally used may be used, and those having a pH of the composition of 5 to 10 are usually preferable. However, a substance that forms an insoluble salt with calcium, such as a phosphate buffer, is not preferable. In addition, those having an amino group end such as trisaminomethane are preferred because they destabilize the PQQ bond of PQQ-dependent glucose dehydrogenase. For this reason, more preferably as a buffering agent, a buffering agent such as boric acid or acetic acid, BES, Bicine, Bis_Tris, CHES, EPPS, HEPES, HEPPSO, MES, MOPS, MOPSO, PIPES, POPS〇, TAPS, TAPS〇, TES Good buffer such as Tricine. Among them, PIPES having a buffer capacity at pH 6 to 7 is preferable. In the powder composition, the content of the buffering agent (W / W) is preferably 1.0% to 50%. More preferably, it is 5% to 10%.

Examples of the amino acid include glycylglycine, dimethyldalycin, histidine, serine, asparagine, aspartic acid, glutamine, and salts thereof, and salts thereof. The amino acid content (WZW) is desirably 10% to 80%, more preferably 20% to 70%, and further preferably 30% to 60%.

Examples of the organic acid include, but are not particularly limited to, ketokenoretaric acid, pyruvic acid, L-malic acid, gnoreclonic acid, and ketokegluconic acid, and salts thereof. The organic acid content (W / W) is desirably 10% to 80%, more preferably 20% to 70%, and further preferably 30% to 60%.

The sugar is not particularly limited, but cyclodextrin and the like are preferable. The saccharide content (WZW) is desirably 10% to 80%, more preferably 20% to 70%, and further preferably 30% to 60%.

The content (W / W) of pyroguchi quinoline quinone is desirably 10% to 80%, more preferably 20% to 70%, and further preferably 30% to 60%.

In addition, a combination of two or more of the above is acceptable. Preferred combinations include those described in FIGS. 1 and 2. The total content (W / W) of the combined substances is desirably 10% to 80%, more preferably 20% to 70%, and even more preferably 30% to 60%.

In addition, as for said reagents, all can use a commercial item.

Furthermore, the present invention is a glucose measurement reagent, a glucose assay kit, and a gnolecose sensor containing the composition. In these, the part relating to the PQQGDH composition can take various forms such as liquid (aqueous solution, suspension, etc.), powdered by vacuum drying or spray drying, freeze drying, and the like. The lyophilization method is not particularly limited and may be performed according to a conventional method. 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.

[0015] glucose assembly kit

The glucose assay kit of the present invention typically has a reagent necessary for measurement such as PQQGDH, buffer solution, and mediator, and a glucose standard for preparing a calibration curve. Includes sub-solutions, as well as guidelines for use. 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 force apoenzyme provided in a holoform, and can be holoed when used.

[0016] 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 immobilization methods 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 may be fixed in a polymer or adsorbed and fixed on an electrode together with an electron mediator typified by mouth sen 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.

[0017] The present invention provides a pyroguchi quinoline quinoline-dependent glucose dehydrogenase composition, which does not contain a protein component other than a host-derived protein component by, for example, the means described above. This is a method for stabilizing quinone-dependent glucose dehydrogenase.

[0018] Furthermore, the present invention provides a stabilized pyroguchi quinoline quinone-dependent gnolecose dehydrogenase characterized by not containing a protein component other than a host-derived protein component by, for example, the means described above. It is a manufacturing method of a composition.

[0019] The PQQ-dependent glucose dehydrogenase activity was measured according to the following method.

50mM

PIPES buffer night, ρΗ 6. 5 25. 5ml, 3. OmM PMS 2. Oml, 6.6mM NTB 1. Om

Contains 1 and 1M glucose 6. 3% TritonX-100

0. Mix 9ml. Incubate this mixture at 37 ° C for 5 minutes, then add the enzyme solution to 0.1 Add ml, mix, and record the change in absorbance at 570 nm for 4 to 5 minutes using a spectrophotometer controlled at 37 ° C with water as a control. Calculate the change in absorbance per minute from the initial linear portion. The amount of enzyme that oxidizes 1 micromole of gnolecose per minute is defined as 1 unit (U).

More specifically, the enzyme activity of PQQGDH can be measured by the following method.

POOGDH ffi †

(1) Measurement principle

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

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: 1 · 0M (1.8g 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 was dissolved in 5N NaOH and 2 ml of 10% Triton X — Calorize 1 00. Use 5N NaOH to 25. Adjust the ρ 6. to 6.5 ± 0.05 with C and add water to make 100 ml.)

C. PMS solution: 3.0mM (9.19mg phenazine methosulfate (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 CaCl, 0.1% Triton X-100, 0.1% BSA included 50mM PIPES—NaOH buffer (ρΗ6 · 5)

Steps

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

1. 0. 9ml D-glucose solution (A)

25. 5ml 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

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. 0.1 ml of enzyme solution was added and mixed by gentle inversion.

4. Record the increase in absorbance for water at 570 nm at 37 ° C for 4-5 minutes with a spectrophotometer and calculate the A OD per minute from the initial linear part of the curve (OD test) The same method was repeated except that the enzyme diluent (E) was added instead of the enzyme solution, and a blank (ΔOD blank) was measured.

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:

U / ml = {Δ OD / min (Δ OD test

Vt: Total volume (3. lml)

Vs: Sample volume (0. lml)

20. 1: 1Z2 mmol molecular extinction coefficient of diformazan

1. 0: Optical path length (cm)

df: dilution factor

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

[0021] The present invention will be specifically described below with reference to examples, but the present invention is not limited by the examples.

Example 1

The following components were dissolved in 6 L of tap water to adjust the pH to 7.0, and then sterilized with an autoclave at 121 ° C. for 20 minutes to obtain a medium.

Glucose 60g Polypeptone 180g Yeast extract 30g NaCl 60g Inoculate 10L jar mentor containing the above medium with Acinetobacter calcoaceticus NCIMB 11517 (National Collection of Industrial Bacte ria, Abadeen Scotland) at 30 ° C After 20 hours of aeration and agitation culture, the cells were collected by centrifugation. The obtained cells are crushed with dynomill, PEI-treated, ammonium sulfate fractionation, phenyl sepharose chromatography and DEAE-sepharose chromatography are purified, and the buffer is replaced with distilled water. Gunolecos dehydrogenase was obtained.

[0022] Example 2

The following compounds were added to the obtained enzyme solution, followed by lyophilization to obtain enzyme powder. After each enzyme powder was stored at 37 ° C for 1 week, the GLD activity was measured to determine the residual activity compared to before storage. The results are shown in Figure 1.

Without using biologically derived substances such as BSA (usi serum albumin) In addition, it was confirmed that organic acids and amino acids showed the same or higher stability.

[0023] Example 3

The following compounds were added to the obtained enzyme solution, followed by lyophilization to obtain enzyme powder. After each enzyme powder was stored at 37 ° C for 1 week, the GLD activity was measured to determine the residual activity compared to before storage. The results are shown in FIG.

It was confirmed that only a buffering agent has a little stabilizing effect, but it is possible to ensure a certain level of stability by adding at least a calcium salt. It was also confirmed that stability could be ensured without adding a conventionally known protein component such as BS A. In addition, even if PQQ was added to the composition consisting of calcium salt and buffer, there was no problem, but a further stabilizing effect was confirmed.

[0024] Example 4

After immobilizing PQQGDH (holo type) of the present invention on a carbon electrode using dartalaldehyde, a glucose sensor is produced by blocking with dartalaldehyde by treatment with a reagent having an amine group. At the time of preparation, both sushi serum albumin (A) and no addition (B) are performed. Next, using the obtained sensor, a 100 mg / dl (concentration) aqueous glucose solution is measured 20 times. Simultaneous reproducibility and accuracy (difference from theoretical values) is better in (B).

Industrial applicability

[0025] According to the present invention, it is possible to provide a PQQ-dependent glucose dehydrogenase excellent in application to a glucose sensor.

Claims

The scope of the claims

[1] A pyroguchi quinoline quinone-dependent gnolecose dehydrogenase composition characterized by not containing a protein component other than a host-derived protein component.

[2] The pyroguchi quinoline quinone-dependent glucose dehydrogenase composition according to claim 1, which contains only calcium or a calcium salt and a buffer in addition to the protein component derived from the host.

[3] The pyroguchi quinoline quinone-dependent glucose dehydrogenase composition according to claim 1, which contains only calcium or calcium salt, pyroguchi quinoline quinone, and a buffer in addition to the host-derived protein component.

[4] Stabilization of pyroguchi quinoline quinone-dependent gnolecose dehydrogenase in a pyroguchi quinoline quinone-dependent gnolecose dehydrogenase composition characterized by not containing a protein component other than the host-derived protein component Method.

[5] In addition to the host-derived protein component, only pyrocalcium quinoline quinone-dependent gnolecose dehydrogenase composition according to claim 4, characterized by containing only calcium or a calcium salt and a buffer. A method for stabilizing oral quinoline quinone-dependent gnolecose dehydrogenase.

[6] A method for producing a stabilized pyroguchi quinoline quinone-dependent gnolecose dehydrogenase composition, which does not contain a protein component other than a host-derived protein component.

[7] The stabilized pyroguchi quinoline quinone-dependent glucose dehydrogenase composition according to claim 6, wherein the composition contains only calcium or a calcium salt and a buffer in addition to the protein component derived from the host. Manufacturing method.

[8] A glucose assembly comprising the glucose dehydrogenase composition according to claim 1.

[9] A glucose sensor comprising the gnolecose dehydrogenase composition according to claim 1.