JP2004236665A - Pqq-dependent glucose dehydrogenase composition and reagent composition for glucose measurement - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a stabilized PQQ-dependent glucose dehydrogenase composition and a reagent composition for measuring glucose using the composition. <P>SOLUTION: Disclosed are a PQQ-dependent glucose dehydrogenase composition containing a calcium ion or calcium salt, and a reagent composition for measuring glucose containing the PQQ-dependent glucose dehydrogenase, a calcium ion or calcium salt, an electron acceptor and buffer solution. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  TECHNICAL FIELD The present invention relates to a stabilized PQQ-dependent glucose dehydrogenase composition and a reagent composition for measuring glucose using the composition.

Conventional methods for measuring glucose in samples such as serum and urine include a chemical method and an enzymatic method, and it is generally said that the enzymatic method is superior in specificity and safety. As an enzymatic method, a glucose oxidase / peroxidase system and a hexokinase / glucose-6-phosphate dehydrogenase system are mainly used, but both measurement systems are not simple reaction systems because a plurality of enzymes are used. In the former (glucose oxidase / peroxidase system), hydrogen peroxide is easily affected by coexisting substances, and it is difficult to use when dissolved oxygen is rate-limiting. The latter (a hexokinase-glucose-6-phosphate dehydrogenase system) requires NAD ⁺ as a coenzyme, and further involves an equilibrium reaction, so that there is a disadvantage that it is difficult to measure a trace amount of glucose.

  The use of a PQQ-dependent glucose dehydrogenase requiring PQQ as a coenzyme in a glucose measurement system is one method for solving the above-mentioned problem of using other enzymes. In particular, when considering measuring glucose using an electric sensor, a system using glucose oxidase is affected by the oxygen concentration, but has an advantage that PQQ-dependent glucose dehydrogenase does not. This is because the enzyme catalyzes the following reaction and catalyzes the reaction of transferring electrons obtained from glucose to an electron acceptor.

  Glucose + electron acceptor → gluconolactone + reduced electron acceptor

  Here, examples of the electron acceptor include ferricyanide, a combination of an electron carrier and 2,6-dichlorophenol indophenol, and a combination of an electron carrier and a tetrazolium salt.

  The PQQ-dependent glucose dehydrogenase used in the present invention is an enzyme that requires PQQ as a coenzyme. For example, Acinetobacter calcoaceticus NCIB11517, Acinetobacter calcoaceticus125 , IFO13006 and the like.

The quantification of glucose using the enzyme is carried out by a method in which PQQ-dependent glucose dehydrogenase is allowed to act on glucose in a sample in the presence of an electron acceptor, and the resulting reduced electron acceptor is measured (eg, non- See Patent Document 1.).
Methods in Enzymology, Vol. 89 (1982) p20 (Academic Press, Inc)

The problem with such a PQQ-dependent glucose dehydrogenase lies in its stability as compared with glucose oxidase and hexokinase, and even when a purified enzyme preparation is added to a clinical test agent, the enzyme is significantly deactivated. Since the reagent is severely deteriorated, it is difficult to store the reagent, and at the same time, the influence on the measured value is large, which is a practical problem. It is said that the addition of PQQ is effective as a stabilizer for the enzyme, but PQQ is very expensive and industrially difficult to add (for example, see Non-Patent Document 2). ).
Arch. Biochem. Biophys. , 218, 623-625 (1982).

  The present inventors have conducted various studies on a stabilizer for PQQ-dependent glucose dehydrogenase. As a result, they have found that the use of calcium ions has a high stabilizing effect, and have reached the present invention.

  That is, the present invention is a PQQ-dependent glucose dehydrogenase composition containing calcium ions or calcium salts.

  Further, the present invention is a glucose measuring reagent composition comprising PQQ-dependent glucose dehydrogenase, calcium ion or calcium salt, and an electron acceptor and a buffer.

  In the present invention, a PQQ glucose dehydrogenase composition that is less expensive and has excellent stability can be obtained. The composition can also be used to accurately measure glucose without being limited by dissolved oxygen.

  In the present invention, the PQQ-dependent glucose dehydrogenase is an enzyme that requires PQQ as a coenzyme. Produced from IFO13006 and the like.

As one of the enzymes, physicochemical properties include, for example, molecular weight: about 55,000 (gel filtration method), Km value: 7.9 × 10 ⁻² M (D-glucose), optimal pH: pH 6 to 7, Those having an optimum temperature of 37 ° C., a pH stability of pH 6 to 7, and a thermal stability of 30 ° C. or less are exemplified.

  In the present invention, the enzyme contains calcium ions or calcium salts. The aqueous composition and the lyophilized product of the enzyme composition of the present invention are not limited as long as they contain these stabilizers.

Examples of the supply form of calcium ions include calcium salts of inorganic or organic acids such as calcium chloride, calcium acetate and calcium citrate. Further, in the aqueous composition, the content of calcium ions is preferably 1 × 10 ⁻⁴ to 1 × 10 ⁻² M.

  In the present invention, preferably, an amino acid selected from the group consisting of glutamic acid, glutamine and lysine can be added in addition to calcium ions or calcium salts. One or more of these amino acids may be used. In the above aqueous composition, the content of the amino acid selected from the group consisting of glutamic acid, glutamine and lysine is preferably 0.01 to 0.2% by weight. When serum albumin is added to the aqueous composition, the content is preferably 0.05 to 0.5% by weight.

  In the present invention, when calcium ions or calcium salts are contained, the effect of improving the stability can be seen, but by containing the amino acid, the stability of glucose dehydrogenase is further increased.

  As the buffering agent, an ordinary one is used, and usually, one having a pH of the composition of 5 to 10 is preferable. Specifically, Tris-HCl, boric acid, and Good's buffer are used, but any buffer that does not form an insoluble salt with calcium can be used.

  If necessary, other components such as a surfactant, a stabilizer, and an excipient may be added to the aqueous composition.

  When preparing the aqueous composition, the order of adding each stabilizer to the buffer is not particularly limited. The freeze-dried product of the present invention is obtained by freeze-drying the aqueous composition according to a usual method.

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

    Mix 25.5 ml of 50 mM PIPES buffer, pH 6.5, 2.0 ml of 3.0 mM PMS, 1.0 ml of 6.6 mM NTB and 0.9 ml of 6.3% Triton X-100 containing 1 M glucose. After incubating the mixture at 37 ° C for 5 minutes, 0.1 ml of the enzyme solution was added and mixed, and the change in absorbance 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 the enzyme that oxidizes 1 micromole of glucose per minute is defined as 1 unit (U).

  The glucose measuring reagent composition of the present invention contains PQQ-dependent glucose dehydrogenase and calcium or a calcium salt, and further contains an electron acceptor and a buffer. Examples of the electron acceptor include ferricyanide, a combination of an electron carrier and 2,6-dichlorophenolindophenol, and a combination of an electron carrier and a tetrazolium salt. In addition, as the buffer, an ordinary buffer is used, and it is generally preferable to adjust the pH of the composition to 5 to 10. Specifically, Tris-HCl, boric acid, and Good's buffer are used, but any buffer that does not form an insoluble salt with calcium can be used.

  As a method for measuring glucose in a sample using the glucose measurement reagent of the present invention, a sample is brought into contact with the glucose measurement reagent of the present invention, and the resulting reduced electron acceptor is subjected to a colorimetric method, an electric sensor, or the like. And the like.

    Hereinafter, the present invention will be specifically described with reference to examples.

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

60g glucose
180 g of polypeptone
Yeast extract 30g
NaCl 60g
A 10 L jar fermenter containing the above-mentioned medium was inoculated with Acinetobacter calcoaceticus NCIB11517 (National Collection of Industrial Bacteria, Abaden Scotland), centrifuged at 30 ° C., centrifuged for 20 hours at 30 ° C., and centrifuged at 30 ° C. for 30 hours. The cells were obtained.

  The obtained cells were disrupted by Dynomill and purified by PEI treatment, ammonium sulfate fractionation, phenyl sepharose chromatography and DEAE-Sepharose chromatography to obtain 600 U of PQQ-glucose dehydrogenase.

  The obtained enzyme was stored in 50 mM Tris-HCl buffer, pH 7.5 containing the following compounds, stored at 30 ° C. for 4 days, and its residual activity was examined. Table 1 shows the obtained results.

Although the addition of 1 mM CaCl ₂ significantly improved the stability of the present invention, the addition of MgSO ₄ had no stabilizing effect. The stability of the present invention was further improved by adding lysine, glutamine or glutamic acid in the presence of 1 mM CaCl ₂ .

The enzyme composition obtained in Example 1 was stored in a 50 mM Tris-HCl buffer containing the following additives, pH 7.5, at 30 ° C for 4 days, and the residual activity was measured. In addition to 1 mM CaCl ₂ , when amino acids which had the effect of further improving the effect of the present invention in Example 1 were further added in combination, there was a more stabilizing effect than when each was used alone. In particular, in the presence of 1 mM CaCl ₂ , the combination with glutamic acid, glutamine or lysine was favorable. On the other hand, it was found that a good stabilizing effect was obtained also when PQQ was added in addition to 1 mM CaCl ₂ . Table 2 shows the results.

PQQ-dependent glucose dehydrogenase was prepared using (1) 50 mM PIPES buffer, pH 7.5, (2) 50 mM PIPES buffer containing 10 mM CaCl ₂ and 0.2% BSA, pH 7.5, (3) 10 mM CaCl ₂ , 0. The thermostability was compared by storing at pH 7.5 in a 50 mM PIPES buffer containing 05% glutamine, 0.05% glutamic acid, 0.05% lysine, 0.2% and BSA. The results are as shown in FIG. 1. The stability of PQQ-dependent glucose dehydrogenase was significantly improved under the condition (2), that is, in a buffer solution containing CaCl ₂ and BSA. Furthermore, the stability of PQQ-dependent glucose dehydrogenase was further improved in the condition (3), that is, in the presence of CaCl ₂ , in a buffer solution in which BSA was combined with glutamine, glutamic acid, and lysine.

5 U / ml of PQQ glucose dehydrogenase of Example 1, 29.5 mM of 1-methoxy-5-phenazolium methyl sulfate, 0.6 mM of MTT, 0.2 mM of NaN ₃ , 10 mM of CaCl ₂ , 0.05% glutamine, 0.05% Glutamic acid, 0.05% lysine, 0.2% BSA, 50 mM PIPES buffer, and pH 7.5 were mixed to obtain a reagent composition for measuring glucose. Using the reagent composition, glucose in serum was measured. The result is as shown in FIG. The absorbance increased linearly with the glucose concentration.

  The PQQ-dependent glucose dehydrogenase composition and the reagent composition for measuring glucose of the present invention can be used for measuring glucose in a sample such as serum or urine. Since the composition of the present invention has excellent stability, glucose measurement can be accurately performed using this composition, which greatly contributes to the industry.

It is a graph which shows the thermostability of PQQ-dependent glucose dehydrogenase. It is a graph which shows a calibration curve of glucose.

Claims (8)

A PQQ-dependent glucose dehydrogenase composition containing calcium ions or calcium salts. The PQQ-dependent glucose dehydrogenase composition according to claim 1, wherein the calcium salt is calcium chloride or calcium acetate or calcium citrate. The PQQ-dependent glucose dehydrogenase composition according to claim 1, wherein the PQQ-dependent glucose dehydrogenase is a PQQ-dependent glucose dehydrogenase derived from Acetobacter calcoaceticus. The PQQ-dependent glucose dehydrogenase composition according to claim 1, further comprising serum albumin. A reagent composition for measuring glucose, comprising a PQQ-dependent glucose dehydrogenase, a calcium ion or a calcium salt, and an electron acceptor and a buffer. The reagent composition for measuring glucose according to claim 5, wherein the calcium salt is calcium chloride, calcium acetate, or calcium citrate. The reagent composition for measuring glucose according to claim 5, wherein the PQQ-dependent glucose dehydrogenase is PQQ-dependent glucose dehydrogenase derived from Acetobacter calcoaceticus. 6. The reagent composition for measuring glucose according to claim 5, further comprising serum albumin.

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