JP2006149378A - Pqq-dependent glucose dehydrogenase composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a glucose assaying system ensuring enzyme stability and excellent in practicability in assaying glucose by a sensor using a PQQ (pyrroloquinoline quinone)-dependent glucose dehydrogenase. <P>SOLUTION: A PQQ-dependent glucose dehydrogenase composition is provided, being characterized by containing no protein component other than host-derived protein components. An alternative PQQ-dependent glucose dehydrogenase composition is provided, containing only calcium or a calcium salt and a buffering agent in addition to host-derived protein components. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a stabilized pyrroloquinoline quinone-dependent glucose dehydrogenase composition. (Hereinafter, pyrroloquinoline quinone is also referred to as PQQ, glucose dehydrogenase as GDH, and pyrroloquinoline quinone-dependent glucose dehydrogenase as PQQGDH, respectively.)

As a method for measuring glucose in samples such as serum and urine, a measurement system using PQQ-dependent glucose dehydrogenase that requires PQQ as a coenzyme is known (for example, Non-Patent Document 1). Although this measurement system can be applied to a reaction in a solution, a glucose sensor is widely used in general (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.
Methods in Enzymology, Vol. 89 (1982) p20 (Academic Press, Inc) Patent Publication 2003-207475

In general, enzyme preparations are added with some kind of stabilizer for the purpose of improving or maintaining the stability thereof, and the same applies to PQQ-dependent glucose dehydrogenase preparations. However, in the case of PQQ-dependent glucose dehydrogenase, if the enzyme is applied to the sensor, the stabilizer may cause unexpected problems such as affecting the measured value and impairing accuracy. Further, in consideration of the solubility of components necessary for the reaction at the time of measurement, it is desirable that the amount of the enzyme preparation having the same activity is small. For these reasons, it is desirable that the added stabilizer is as small as possible, and it is desirable to ensure stability and accuracy as an enzyme preparation and further as a sensor.
The subject of this invention is to make compatible the stability of an enzyme formulation, and the reduction of the factor which impairs the accuracy of a measurement when it applies to a sensor.

As a result of diligent studies on the stabilizer for PQQ-dependent glucose dehydrogenase, the present inventor has found a composition that can ensure stability without using a biological substance, and has reached the present invention.

The outline of the present invention is as follows. That is,
[Claim 1]
A pyrroloquinoline quinone-dependent glucose dehydrogenase composition comprising no protein component other than a host-derived protein component.
[Section 2]
The pyrroloquinoline quinone-dependent glucose dehydrogenase composition according to claim 1, comprising only calcium or a calcium salt and a buffer in addition to the protein component derived from the host.
[Section 3]
The pyrroloquinoline quinone-dependent glucose dehydrogenase composition according to claim 1, comprising only calcium or calcium salt, pyrroloquinoline quinone, and a buffer in addition to the host-derived protein component.
[Claim 4]
A method for stabilizing pyrroloquinoline quinone-dependent glucose dehydrogenase in a pyrroloquinoline quinone-dependent glucose dehydrogenase composition, which does not contain a protein component other than a host-derived protein component.
[Section 5]
The pyrroloquinoline quinone-dependent glucose dehydrogenase in the pyrroloquinoline quinone-dependent glucose 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. Stabilization method.
[Claim 6]
A method for producing a stabilized pyrroloquinoline quinone-dependent glucose dehydrogenase composition, which does not contain a protein component other than a host-derived protein component.
[Claim 7]
The method for producing a stabilized pyrroloquinoline 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 assay kit comprising the glucose dehydrogenase composition according to claim 1.
[Claim 9]
A glucose sensor comprising the glucose dehydrogenase composition according to claim 1.

  According to the present invention, it is possible to provide a glucose measurement system that ensures the stability of the enzyme and is highly practical in measuring glucose with a sensor using a PQQ-dependent glucose dehydrogenase. In addition, it is possible to provide a glucose measurement system having both practical stability and measurement accuracy and excellent practicality.

In the present invention, PQQ-dependent glucose dehydrogenase is an enzyme that coordinates PQQ as a coenzyme, and an enzyme that catalyzes the reaction of oxidizing D-glucose to produce D-glucono-1,5-lactone ( EC 1.1.5.2 (formerly EC 1.1.9.17)), and the origin and structure are not particularly limited.
For example, microorganisms are produced from Acinetobacter genus bacteria, Pseudomonas genus bacteria, Burkholderia genus bacteria, Gluconobacter genus bacteria, or Acetobacter genus bacteria.
For example, Acinetobacter baumannii (see, for example, Patent Document 1), Acinetobacter calcoaceticus (see, for example, Non-Patent Documents 1 and 2), Pseudomonas aeru in a eu ag au g. Oxidative bacteria such as Pseudomonas putida, Pseudomonas fluorescens, Gluconobacter oxydans (see, for example, Non-Patent Document 3) and Agrobacterium radiobacterium (Agrobacterium radio acter), Escherichia coli (see, for example, Non-Patent Document 4), enterobacteria such as Klebsiella aerogenes, and Burkholderia cepacia. it can.
Moreover, it is produced from, for example, Acinetobacter baumannii NCIMB11517 strain, Acinetobacter calcaceticus IFO12552 strain, Acinetobacter calcaceticus LMD79.41 strain, etc. (Patent Documents 2, 3, 4).
A. M.M. Cleton-Jansen et al. Bacteriol. , 170, 2121 (1988) Mol. Gen. Genet. , 217, 430 (1989) Mol. Gen. Genet. , 229, 206 (1991) A. M.M. Cleton-Jansen et al. Bacteriol. , 172, 6308 (1990) JP-A-11-243949 JP 2004-173538 A Special table 2004-512047

PQQGDH that can be applied to the present invention may have some other amino acid residues deleted or substituted with those exemplified above as long as it has glucose dehydrogenase activity, and other amino acid residues may be removed. A group 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 a base sequence of a gene encoding a protein in order to introduce a site-specific mutation into the protein are described in Sambrook et al., Molecular Cloning; A Laboratory Manual 2nd edition (1989). Cold Spring Harbor Laboratory Press, New York.

These PQQGDHs can be easily produced by those skilled in the art using known techniques in the art.
For example, the above-mentioned natural microorganisms producing PQQGDH, or the gene encoding natural PQQGDH as it is or after being mutated, are expressed vectors (many are known in the art. For example, plasmids ), And cultivates the transformant transformed into an appropriate host (many are known in the art. For example, E. coli), and recovers the cell from the culture by centrifugation. After that, the bacterial 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 solubilized, and a water-soluble fraction containing PQQGDH. You can get minutes. 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 precipitated by, for example, vacuum concentration, 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, etc. You just have to let them know. Heat treatment and isoelectric point treatment are also effective purification means. Further, purified PQQGDH can be obtained by performing gel filtration with an adsorbent or a gel filter, adsorption chromatography, ion exchange chromatography, and affinity chromatography. The purified enzyme preparation is preferably purified to the extent that it shows a single band on electrophoresis (SDS-PAGE).
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.

PQQGDH produced as described above substantially does not contain protein components other than the host-derived protein components.
Alternatively, for example, a commercially available enzyme such as GLD-321 manufactured by Toyobo Co., Ltd. can be similarly purified to produce PGGGDH containing no protein component other than the host-derived protein component.

The composition of the present invention can be obtained by passing through a process other than “adding a host-derived protein” to PQQGDH containing no protein component other than the host-derived protein component produced as described above. it can. For example, nothing may be added to PQQGDH, and it can be obtained by adding various components other than the host-derived protein component.
The form of the composition can take various forms such as an aqueous composition such as a 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, and may be in a solution state in which the lyophilized product is redissolved.

Alternatively, the composition of the present invention basically consists of the enzyme and calcium or calcium salt and a buffer. Moreover, after containing these, pyrroloquinoline quinone, amino acid, saccharide or organic acid may be further added. 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 supply form of calcium include calcium salts of inorganic acids or organic acids such as calcium chloride, calcium acetate, and calcium citrate. 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%.
Any buffering agent that is generally used may be used, and usually the buffering agent having a pH of 5 to 10 is preferable. However, those that form an insoluble salt with calcium such as a phosphate buffer are not preferred. Those having an amino group end such as trisaminomethane are not preferable because the PQQ bond of PQQ-dependent glucose dehydrogenase is unstable. 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, POPSO, TAPS, TAPSO, TES Good buffer such as Tricine. Among these, PIPES having a buffer capacity at pH 6 to 7 is preferable. Further, in the powder composition, the content (W / W) of the buffering agent is desirably 1.0% to 50%, and more preferably 5% to 10%.
The amino acid is not particularly limited, and preferred examples include glycylglycine, dimethylglycine, histidine, serine, asparagine, aspartic acid, glutamine, and salts thereof. The amino acid content (W / W) is desirably 10% to 80%, more preferably 20% to 70%, and further preferably 30% to 60%.
Although it does not specifically limit as an organic acid, (alpha) ketoglutaric acid, pyruvic acid, L-malic acid, glucuronic acid, alpha ketogluconic acid etc. or those salts are mentioned as a preferable thing. The organic acid content (W / W) is desirably 10% to 80%, more preferably 20% to 70%, and further preferably 30% to 60%.
Although it does not specifically limit as saccharides, alpha cyclodextrin etc. are mentioned as a preferable thing. The saccharide content (W / W) is desirably 10% to 80%, more preferably 20% to 70%, and further preferably 30% to 60%.
The content (W / W) of pyrroloquinoline quinone is desirably 10% to 80%, more preferably 20% to 70%, and further preferably 30% to 60%.
Further, a combination of two or more of the above may be used. Preferred combinations include those described in FIGS. 1 and 2. The total content (W / W) of the combined materials is desirably 10% to 80%, more preferably 20% to 70%, and further 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 glucose sensor containing the composition. In these, the part which concerns on a PQQGDH composition can take various forms, such as liquid (aqueous solution, suspension, etc.), what was pulverized by vacuum drying, spray drying, etc., freeze drying. 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, and may be in a solution state in which the lyophilized product is redissolved.

Glucose Assay Kit The glucose assay kit of the present invention typically comprises reagents necessary for measurement such as PQQGDH, buffer solution, mediator, glucose standard solution for creating a calibration curve, and usage guidelines. The kit of the 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 a holified form, but may be provided in the form of an apoenzyme and holified at the time of use.

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 photocrosslinkable polymer, a conductive polymer, a redox polymer, or the like, or ferrocene or It may be fixed in a polymer or adsorbed and fixed on an electrode together with an electron mediator typified by a derivative thereof, or may be used in combination. Preferably, the PQQGDH of the present invention is immobilized on the electrode in a holo form, but it is also possible to immobilize it in the form of an apoenzyme and supply PQQ as a separate layer or in solution. Typically, 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 present invention provides the stability of pyrroloquinoline quinone-dependent glucose dehydrogenase in a pyrroloquinoline quinone-dependent glucose dehydrogenase composition characterized by not containing a protein component other than a host-derived protein component by, for example, the means described above. It is a conversion method.

  Further, the present invention is a method for producing a stabilized pyrroloquinoline quinone-dependent glucose dehydrogenase composition characterized in that no protein component other than a host-derived protein component is contained, for example, by the means described above. .

  The PQQ-dependent glucose dehydrogenase activity was measured according to the following method. Mix 0.9 ml of 50 mM PIPES buffer, pH 6.5 25.5 ml, 3.0 mM PMS 2.0 ml, 6.6 mM NTB 1.0 ml and 1% glucose 6.3% Triton X-100. After incubating this mixed solution at 37 ° C. for 5 minutes, 0.1 ml of enzyme solution was added, and after mixing, the absorbance change at 570 nm was recorded for 4 to 5 minutes using a spectrophotometer controlled at 37 ° C. with water as a control. The change in absorbance per minute is determined from the initial linear portion. The amount of enzyme that oxidizes 1 micromole of glucose per minute is defined as 1 unit (U).

More specifically, the enzyme activity of PQQGDH can be measured by the following method.
Method for measuring PQQGDH enzyme activity (1) Principle of measurement D-glucose + PMS + PQQGDH → D-glucono-1,5-lactone + PMS (red)
The presence of diformazan formed by the reduction of nitrotetrazolium blue (NTB) by 2PMS (red) + NTB → 2PMS + diformazan phenazine methosulfate (PMS) (red) was measured spectrophotometrically at 570 nm.
(2) Definition of Unit 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: 1.0 M (1.8 g D-glucose (molecular weight 180.16) / 10 ml H2O)
B. PIPES-NaOH buffer, pH 6.5: 50 mM (1.51 g of PIPES (molecular weight 302.36) suspended in 60 mL of water is dissolved in 5N NaOH and 2.2 ml of 10% Triton X-100 is added. The pH was adjusted to 6.5 ± 0.05 at 25 ° C. using 5N NaOH, and water was added to make 100 ml.)
C. PMS solution: 3.0 mM (9.19 mg phenazine methosulfate (molecular weight 817.65) / 10 ml H2O)
D. NTB solution: 6.6 mM (53.96 mg of nitrotetrazolium blue (molecular weight 817.65) / 10 ml H2O)
E. Enzyme dilution: 50 mM PIPES-NaOH buffer (pH 6.5) containing 1 mM CaCl ₂ , 0.1% Triton X-100, 0.1% BSA
Procedure Prepare the following reaction mixture in a light-proof bottle and store on ice (prepared at the time of use)
1. 0.9 ml D-glucose solution (A)
25.5 ml PIPES-NaOH buffer (pH 6.5) (B)
2.0ml PMS solution (C)
1.0ml NTB solution (D)

The concentrations in the assay mixture are as follows:
PIPES buffer 42 mM
D-glucose 30 mM
PMS 0.20 mM
NTB 0.22mM

2. 3.0 ml of the reaction mixture was placed in a test tube (made of plastic) and preheated at 37 ° C. for 5 minutes.
3. Add 0.1 ml enzyme solution and mix by gentle inversion.
The increase in absorbance for water at 4.570 nm was recorded on a spectrophotometer while maintaining at 37 ° C. for 4-5 minutes and the ΔOD 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 the enzyme diluent (E) was added instead of the enzyme solution.
Immediately before the assay, the enzyme powder was dissolved in an ice-cold enzyme diluent (E) and diluted to 0.1-0.8 U / ml with the same buffer (use of a plastic tube for adhesion of the enzyme) Is preferred).
Calculation Activity is calculated using the following formula:
U / ml = {ΔOD / min (ΔOD test−ΔOD blank) × Vt × df} / (20.1 × 1.0 × Vs)
U / mg = (U / ml) × 1 / C
Vt: Total volume (3.1 ml)
Vs: Sample volume (0.1 ml)
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)

EXAMPLES Hereinafter, although an Example is given and this invention is shown concretely, this invention is not limited by an Example.
Example 1
The following components were dissolved in 6 L of tap water and the pH was adjusted to 7.0, and then sterilized with an autoclave at 121 ° C. for 20 minutes to obtain a medium.
Glucose 60 g Polypeptone 180 g Yeast extract 30 g NaCl 60 g The 10 L jar fermenter containing the above medium was inoculated with Acinetobacter calcoaceticus NCIMB 11517 (National Collection of Industrial Bacterium 30 ° C.) After stirring culture, the cells were obtained by centrifugation. The obtained cells were crushed with dynomill, subjected to PEI treatment, ammonium sulfate fractionation, purification by phenyl sepharose chromatography and DEAE-sepharose chromatography, and buffer replacement with distilled water, and 600 U of PQQ-dependent glucose dehydrogenase Got.

Example 2
The following compound was 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 with that before storage. The results are listed in FIG.
It has been confirmed that organic acids and amino acids exhibit the same or higher stability without using a conventionally derived biological material such as BSA (bovine serum albumin).

Example 3
The following compound was 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 with that before storage. The results are shown in FIG.
Although the stabilizing effect is small with only the buffer, it was confirmed that a certain level of stability can be ensured by adding at least a calcium salt. It has also been confirmed that stability can be ensured without adding a conventionally known protein component such as BSA. It should be noted that there was no problem even if PQQ was added to the composition composed of the calcium salt and the buffering agent, but a further stabilizing effect was confirmed.

Example 4
The PQQGDH (holo type) 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 to produce a glucose sensor. At the time of preparation, both bovine serum albumin addition (A) and no addition (B) are performed. Next, a 100 mg / dl (concentration) aqueous glucose solution is measured 20 times using the obtained sensor. Simultaneous reproducibility and accuracy (difference from theoretical values) are superior to (B).

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

Comparison of glucose dehydrogenase residual activity after storage in each composition of Example 2 Comparison of glucose dehydrogenase residual activity after storage in each composition of Example 3

Claims (9)

  A pyrroloquinoline quinone-dependent glucose dehydrogenase composition comprising no protein component other than a host-derived protein component.   The pyrroloquinoline quinone-dependent glucose dehydrogenase composition according to claim 1, comprising only calcium or a calcium salt and a buffer in addition to the protein component derived from the host.   The pyrroloquinoline quinone-dependent glucose dehydrogenase composition according to claim 1, comprising only calcium or calcium salt, pyrroloquinoline quinone, and a buffer in addition to the host-derived protein component.   A method for stabilizing pyrroloquinoline quinone-dependent glucose dehydrogenase in a pyrroloquinoline quinone-dependent glucose dehydrogenase composition, which does not contain a protein component other than a host-derived protein component.   The pyrroloquinoline quinone-dependent glucose dehydrogenase in the pyrroloquinoline quinone-dependent glucose dehydrogenase composition according to claim 4, which contains only calcium or a calcium salt and a buffer in addition to the protein component derived from the host. Stabilization method.   A method for producing a stabilized pyrroloquinoline quinone-dependent glucose dehydrogenase composition, which does not contain a protein component other than a host-derived protein component.   The method for producing a stabilized pyrroloquinoline 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.   A glucose assay kit comprising the glucose dehydrogenase composition according to claim 1.   A glucose sensor comprising the glucose dehydrogenase composition according to claim 1.

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