JP3041839B2 - Method for stabilizing phosphoenolpyruvate carboxylase, stabilizing agent, and stabilized composition for measuring carbon dioxide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION Phosphoenolpyruvate carboxylase of the present invention (EC.4.1.1.13 or less PEP)
(Hereinafter abbreviated as Case) and PEPCase.

[0002] The blood of higher organisms is constantly regulated within a certain pH range by homeostasis, but it is known that in certain diseases this pH tends to be abnormally acidic or alkaline. Such changes in blood pH are caused by changes in the balance of each ion such as sodium, potassium, phosphate, and carbonate in body fluids, and the amount of carbon dioxide in the blood is measured to understand the disease state. To do is clinically very significant.

[0003] Methods for measuring carbon dioxide include a method using an electrode and a method using an enzyme, but the enzyme method is advantageous because a special detection device is not required and a large number of samples can be measured in a short time. . PEPCase converts carbon dioxide into phosphoenolpyruvate (hereinafter referred to as PEP) as shown in Chemical Formula 1 below.
(Hereinafter abbreviated as) and catalyzes the reaction to produce oxaloacetic acid, which is used in an enzymatic measurement method of carbon dioxide using a reaction system shown in Chemical formula 2 below.

[0004]

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[0005]

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[0006] PEPCase is widely distributed in the natural world and is particularly famous for those derived from C4 plants such as corn. However, those derived from microorganisms such as C3 plants, cyanobacteria, Escherichia coli and Pseudomonas bacteria are known. Reports have been made [The Enzyme (3rd ed.),
6, p. 117-168 (1972), Plant P
hysiol. , 57, 906-910 (1976),
B. B. R. C. , 133, 436-441 (198).
5) etc.].

In general, the stable pH range of this enzyme is weakly acidic to neutral regardless of its origin, and rapidly deactivates on the alkaline side. However, since the carbon dioxide to be measured is scattered in a weak acid, this enzyme is actually used only for p.
The weak alkaline property of H7.5-8.0, and the poor stability of PEPCase in the reaction reagent was one of the major problems in the measurement of carbon dioxide by the enzymatic method.

Conventionally, many studies have been made to improve the stability of PEPCase. For example, addition of aspartic acid / ammonium sulfate / EDTA [The Enzyme
(3rd ed.), 6, p117-168 (197)
2), U.S. Pat. No. 3,963,578] and the addition of a protease inhibitor (JP-A-1-95779). However, these stabilizers are unsuitable for being added to the reagent for measuring carbon dioxide, and the stability on the alkaline side of PEPCase may not always be greatly improved even by the addition of the stabilizer, The life of the enzyme in the reaction reagent was extremely short.

[0009]

Generally, non-volatile organic solvents such as polyhydric alcohols and sugars / sugar alcohols have a function of lowering the polarity of a solution, and impede the salting-out effect of salts such as ammonium sulfate. There is no example used for the purpose of improving stability. In addition, there is no description in the references described above that ammonium sulfate and an organic solvent or sugar / sugar alcohol coexist, and there is an example of adding glycerol for the purpose of improving reactivity. Is [P
lant Physiol. , 57, 906-910.
(1976)], does not coexist with ammonium sulfate, and does not mention any improvement in stability.

The present inventors have made various studies on means for stabilizing PEPCase without substantially lowering the performance of the reagent for measuring carbon dioxide, and as a result, in the presence of ammonium sulfate, a non-volatile compound containing an oxygen atom in the molecule. PEPCase by adding a neutral organic solvent and / or a sugar / sugar alcohol
Stability is dramatically improved not only in the weakly acidic to neutral range but also in the weakly alkaline range,
The present invention has been completed.

[0011]

The gist of the present inventor is as follows.
(1) PEP characterized by coexisting (a) ammonium sulfate, (b) a non-volatile organic solvent containing an oxygen atom in a molecule, and / or (c) a sugar or a sugar alcohol with PEPCase.
Case stabilization method, (2) (a) ammonium sulfate, (b) non-volatile organic solvent containing oxygen atom in molecule and / or (c)
A stabilizer of phosphoenolpyruvate carboxylase characterized by containing a sugar or a sugar alcohol; and (3) phosphoenolpyruvate, reduced coenzyme, P
In a composition for measuring carbon dioxide comprising EPCase and malate dehydrogenase, (a) ammonium sulfate, (b) a non-volatile solvent containing an oxygen atom in the molecule, and / or (c) sugar or sugar alcohol coexist. A stabilized composition for measuring carbon dioxide.

The non-volatile organic solvent containing an oxygen atom in the molecule as referred to in the present invention includes polyhydric alcohols such as glycerol and ethylene glycol and their polycondensates and derivatives thereof, dimethyl sulfoxide, ether compounds, ester compounds and the like. And the like.

The sugars include monosaccharides such as mannose and galactose, disaccharides such as sucrose and trehalose, and oligosaccharides such as salicin. Sugar alcohols include inositol, mannitol, sorbitol, and the like.
It refers to glucitol and the like.

In practicing the present invention, the PEP used
As the case, a commercially available corn-derived enzyme or wheat-derived enzyme can be used, or it can be prepared by purification from a culture of blue-green algae or a microorganism. The enzyme concentration can be dissolved to any concentration set by the practitioner, but according to the method of the present invention, PEPCase is 0.1 U
The effect can be shown even in an enzyme-diluted solution of not more than / ml. However, the invention does not limit the source or presence of PEPCase in any way.

In the present invention, the stabilizer added for stabilizing the enzyme is conventionally frequently used as a coexisting substance of the enzyme, and when used alone or in combination, adversely affects other enzymes in the reagent for measuring carbon dioxide. And is suitable for measurement with an automatic analyzer.

The amount of the stabilizer can be adjusted within a range that does not cause inconvenience in handling the reagent containing the enzyme. For example, the penetration of the measuring reagent such as 1% (w / v) or less for both ammonium sulfate and the organic solvent can be adjusted. The effect can be exhibited at a low concentration that does not significantly increase the pressure and viscosity. The fact that the effect is obtained at such a low concentration is important in suppressing the daily variation and the daily variation of the measured value not only in the method of use but also in the case of measurement with an automatic analyzer.

The type of the buffer used in the present invention and the concentration thereof are not particularly limited, but the pH is 5.5 to 8.5.
It is desirable that the concentration is set to a value that has a buffer capacity between the two and maintains a necessary and sufficient buffer capacity. A general-purpose phosphate buffer or Tris buffer can be used as such a buffer, and BES, HEPES, TE
A good buffer such as S can also be used. The buffer concentration is preferably 10 mM to 0.5 M, more preferably 50 mM to 0.1 M. Further, the buffer may contain a chelating agent, a sulfhydryl compound, an inorganic salt, bovine serum albumin, an amino acid, a surfactant, an antibiotic, and the like.

In order to prevent the carbon dioxide in the air from dissolving in the carbon dioxide measurement reagent and consuming the reduced coenzyme and PEP, the container storing the reagent is sealed,
It is a common method to perform treatments such as nitrogen aeration and addition of a carbonate ion absorber.

[0019]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto. Example 1 Corn corn-derived PEPCase was dissolved at a concentration of 0.2 U / ml in the presence of 5.0% (w / v) of each compound in a 50 mM Tris-HCl buffer containing 1.0% (w / v) ammonium sulfate. . This is stored at 25 ° C and PEPCase
Was tracked for residual activity. The results clearly show that a high stabilizing effect is obtained by the coexistence of each compound, as shown in Table 1. Table 1 Additives Residual rate (after 8 days) None 15 (%) Glycerol 50 Ethylene glycol 48 Dimethyl sulfoxide 36 Inositol 28 Sorbitol 33

Example 2 5.0% (w / v) of each compound was added to 50 mM Tris-HCl buffer containing 1.0% (w / v) of ammonium sulfate to make wheat-derived PEPCase 0.2 U / ml. Dissolved. This was stored at 25 ° C. and the residual activity of PEPCase was monitored. As shown in Table 2, it is clear that a high stabilizing effect was obtained by the coexistence of each compound. Table 2 Additives Residual rate (after 8 days) None 14 (%) Glycerol 48 Ethylene glycol 60 Dimethyl sulfoxide 45 Inositol 47 Sorbitol 40 Saccharose 31

Example 3 Corn-derived PEPCase was dissolved in 0.1 M BES buffer, pH 8.0, and the amounts of ammonium sulfate and glycerol or ethylene glycol were changed.
And the remaining activity was followed. Table 3 shows the results. As is clear from Table 3, the stabilizing effect of ammonium sulfate and each compound is increased by increasing the concentration thereof.
% And the effect is still exhibited. Table 3 Concentration (%) Residual rate (%) Ammonium sulfate Glycerol Ethylene glycol Day 4 Day 7 0 16 3 0.5 0.1 88 42 1.0 0.5 98 98 68 1.5 5.0 98 77 0 0.5 0.1 57 32 1.0 0.5 73 45 1.0 1.0 86 86 66 1.5 10.0 88 76

Example 4 A methanol-assimilating microorganism A microorganism belonging to the genus Hypomicrobium was cultured, and PEPCase was prepared from the culture. This PEPCase is dissolved in a 50 mM HEPES buffer containing 1.5% ammonium sulfate and 5% ethylene glycol, pH 7.3 to a concentration of 0.05 U / ml, and stored at 25 ° C. to track the residual activity of the enzyme. did.

Comparative Example 1 PEPC derived from a microorganism belonging to the genus Hypomicrobium
50 mM HE so as to be 0.05 U / ml.
It was dissolved in PES buffer, pH 7.3, stored at 25 ° C., and the remaining activity of the enzyme was monitored.

Comparative Example 2 PEPC derived from a microorganism belonging to the genus Hypomicrobium
ASE was dissolved in 50 mM HEPES buffer (pH 7.3) containing 1.5% ammonium sulfate to a concentration of 0.05 U / ml, and the solution was stored at 25 ° C. and the remaining activity of the enzyme was monitored.

Comparative Example 3 PEPC derived from a microorganism belonging to the genus Hypomicrobium
50 mM HEPES buffer containing 5% ethylene glycol so that
The enzyme was dissolved in H7.3 and stored at 25 ° C., and the remaining activity of the enzyme was monitored. Table 4 also shows the results of Example 4 and Comparative Examples 1 to 3. From these results, it is clear that there is a strong synergistic effect between ammonium sulfate and ethylene glycol.

[0026]

Example 5 To remove the dissolved carbon dioxide, a degassed and aerated nitrogen was added to a 0.1 M HEPES buffer containing 1% ammonium sulfate and 1.5% glycerol, pH 7.5. 10 mM phosphoenolpyruvate, 1.5 U / ml phosphoenolpyruvate carboxylase, 0.5 mM reduced nicotinamide adenine dinucleotide, 2 U / ml malate dehydrogenase, 1 mM magnesium chloride, 0.1 mM ethylenediaminetetraacetate And dissolved to prepare a reagent solution for measuring carbon dioxide. The test solution was aerated with nitrogen, sealed and stored at 25 ° C. for 3 days, and a calibration curve was prepared using potassium hydrogen carbonate as a sample.

Comparative Example 4 Example 5 using 0.1 M HEPES buffer, pH 7.5, but without ammonium sulfate and glycerol.
In the same manner as described above, a test solution for measuring carbon dioxide was prepared. After treating this test solution in the same manner as in Example 5, a calibration curve was prepared using potassium hydrogen carbonate as a sample. FIG. 1 shows Example 5 and Comparative Example 4.
Are also shown. From this result, it is understood that the stability of the test solution for measuring carbon dioxide is improved by the present invention.

[0029]

According to the present invention, the stability of PEPCase is greatly improved, and a stabilized composition for measuring carbon dioxide is provided.

[Brief description of the drawings]

FIG. 1 is a calibration curve prepared using potassium bicarbonate as a sample for a carbon dioxide measuring reagent of the present invention and a carbon dioxide measuring reagent containing no stabilizer.

[Explanation of symbols]

 1: Reagent for measuring carbon dioxide of the present invention 2: Reagent for measuring carbon dioxide not containing a stabilizer

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1/25-1/66 C12N 9/00-9/99 CA (STN) BIOSIS (DIALOG) WPI (DIALOG) )

Claims (3)

(57) [Claims] 1. A phosphoenol characterized in that (a) ammonium sulfate, (b) a non-volatile organic solvent containing an oxygen atom in a molecule and / or (c) a sugar or a sugar alcohol coexist with phosphoenol pyruvate carboxylase. A method for stabilizing pyruvate carboxylase. 2. A stabilizer for phosphoenolpyruvate carboxylase, comprising (a) ammonium sulfate, (b) a nonvolatile organic solvent containing an oxygen atom in the molecule and / or (c) a sugar or a sugar alcohol. . 3. A composition for measuring carbon dioxide comprising phosphoenolpyruvate, reduced coenzyme, phosphoenolpyruvate carboxylase and malate dehydrogenase, wherein (a) ammonium sulfate, and (b) oxygen in the molecule. A stabilized composition for measuring carbon dioxide, characterized by coexisting a non-volatile organic solvent containing atoms and / or (c) sugar or sugar alcohol.