JP7294319B2 - Method for stabilizing ascorbate oxidase - Google Patents

Description

The present invention relates to a method for stabilizing ascorbate oxidase.

Various methods for enzymatically measuring biological components have been developed so far. Among these methods, the method using an oxidase such as cholesterol oxidase introduces a peroxide produced by the action of the oxidase into a color-developing system of a hydrogen donor in the presence of peroxidase for colorimetric determination. Since this measurement system is easily affected by ascorbic acid in biological components, ascorbic acid oxidase (hereinafter sometimes abbreviated as ASO) is added to ascorbic acid present in biological components before or at the time of hydrogen peroxide measurement. It is common practice to react and convert to dehydroascorbic acid to avoid the effects. However, ASO in a solution is very unstable and deactivates during storage of the reagent, which is a factor affecting the storage stability of the reagent.

On the other hand, it has been shown that the use of catalase, gelatin, globulin, peroxidase, methemoglobin, hematin, or the like as a stabilizer is effective. Further, it is disclosed that the storage stability of ASO is improved by adding a compound comprising a metal and an anionic acid group and catalase and/or peroxidase (see, for example, Patent Document 1). Also, a method of adding a sugar alcohol to a solution containing ascorbic acid oxidase (see, for example, Patent Document 2), a method of allowing nitrous acid or a salt thereof, or a nitrite ester to coexist in an aqueous medium (see, for example, Patent Document 3), A method using a substance selected from 2-methyl-4-isothiazolin-3-one, 5-chloro-2-methyl-4-isothiazolin-3-one, 1,2-benzisothiazolin-3-one and salts thereof ( For example, see Patent Document 4) and a method of blending bilirubic acid (salt) (see Patent Document 5, for example) are disclosed. However, these methods still have problems in terms of tolerable stability as liquid reagents.

Japanese Patent Publication No. 8-15430 JP-A-2003-116539 WO2013/176225 Japanese Patent No. 4288559 Japanese Patent No. 4620881

The problem to be solved by the present invention is to provide a method for stabilizing ASO so that it can be used as a stable liquid reagent for a long period of time.

The present inventors have made intensive studies to achieve the above object, and found that ASO can be kept stable in a liquid state by allowing ASO to coexist with a certain surfactant. coexisting with a combination of the surfactant and a certain buffering agent, the ASO can be more effectively kept stable in a liquid state, and the present invention has been completed. That is, the present invention consists of the following configurations.

[Item 1] Ascorbic acid oxidase, polyoxyethylene distyrenated phenyl ether, polyoxyethylene tribenzylphenyl ether, polyoxyethylene lauryl ether, polyoxyethylene alkyl ether, polyoxyalkylene alkyl ether, and polyoxyethylene nonylphenyl A method for stabilizing ascorbate oxidase, which comprises coexisting with at least one surfactant selected from the group consisting of ethers.
[Item 2] The surfactant is at least one interface selected from the group consisting of Emulgen A60, Emulgen A90, Emulgen B66, Emulgen 108, Emulgen 120, Emulgen 707, Emulgen LS110, Emulgen LS114, and nonionic NS-210. Item 2. The method of Item 1, which is an active agent.
[Item 3] Ascorbic acid oxidase, polyoxyethylene distyrenated phenyl ether, polyoxyethylene tribenzylphenyl ether, polyoxyethylene lauryl ether, polyoxyethylene alkyl ether, polyoxyalkylene alkyl ether, and polyoxyethylene nonylphenyl A method for stabilizing ascorbate oxidase, which comprises coexisting with a buffer solution containing at least one surfactant selected from the group consisting of ethers and an amine.
[Item 4] The surfactant is at least one interface selected from the group consisting of Emulgen A60, Emulgen A90, Emulgen B66, Emulgen 108, Emulgen 120, Emulgen 707, Emulgen LS110, Emulgen LS114, and nonionic NS-210. Item 4. The method of Item 3, which is an active agent.
[Item 5] The buffer solution containing the amine is N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N-(2-acetamido)iminodiacetic acid (ADA), N,N-bis(2 -hydroxyethyl)-2-aminoethanesulfonic acid (BES), N,N-bis(2-hydroxyethyl)glycine (Bicine), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (EPPS), 2-[4-(2-hydroxyethyl)-1 -piperazinyl]ethanesulfonic acid (HEPES), 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPSO), 2-morpholinoethanesulfonic acid, monohydrate (MES ), 3-morpholinopropanesulfonic acid (MOPS), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), piperazine-1,4- Bis(2-hydroxy-3-propanesulfonic acid), dihydrate (POPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-tris(hydroxymethyl)methyl-2 Item 3 or Item 3, which is at least one selected from the group consisting of -aminoethanesulfonic acid (TES), N-[tris(hydroxymethyl)methyl]glycine (Tricine) and tris(hydroxymethyl)aminomethane (Tris) 5. The method for stabilizing ascorbate oxidase according to 4.
[Item 6] from the group in which the amine-containing buffer solution consists of N-(2-acetamido)iminodiacetic acid (ADA) and N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) Item 6. The method for stabilizing ascorbate oxidase according to any one of Items 3 to 5, which is at least one selected.
[Item 7] Ascorbate oxidase and a composition in which ascorbate oxidase is stabilized, comprising the following (1).
(1) selected from the group consisting of polyoxyethylene distyrenated phenyl ether, polyoxyethylene tribenzylphenyl ether, polyoxyethylene lauryl ether, polyoxyethylene alkyl ether, polyoxyalkylene alkyl ether, and polyoxyethylene nonylphenyl ether; at least one surfactant.
[Item 8] A composition in which ascorbate oxidase is stabilized, comprising ascorbate oxidase, the following (1) and the following (2).
(1) selected from the group consisting of polyoxyethylene distyrenated phenyl ether, polyoxyethylene tribenzylphenyl ether, polyoxyethylene lauryl ether, polyoxyethylene alkyl ether, polyoxyalkylene alkyl ether, and polyoxyethylene nonylphenyl ether; at least one surfactant,
(2) buffers containing amines;
[Item 9] A biological component analysis kit, comprising the composition according to Item 7 or Item 8.

According to the present invention, a stable ASO-containing liquid preparation can be provided, and accurate measurement values can be obtained without being affected by ascorbic acid even after storage for a long period of time or under severe conditions.

Detailed descriptions of the embodiments of the present invention are as follows, but the present invention is not limited thereto. All non-patent documents and patent documents described in this specification are incorporated herein by reference. In the present specification, "~" means "more than or equal to or less than". For example, "X to Y" in the specification means "X or more and Y or less". Also, "and/or" in this specification means either or both. Also, in this specification, it should be understood that expressions in the singular also include the concept of the plural unless stated otherwise.

(Stabilization of ascorbate oxidase by surfactant)
One embodiment of the present invention provides ascorbate oxidase with polyoxyethylene distyrenated phenyl ether, polyoxyethylene tribenzylphenyl ether, polyoxyethylene lauryl ether, polyoxyethylene alkyl ether, polyoxyalkylene alkyl ether, and at least one surfactant selected from the group consisting of polyoxyethylene nonylphenyl ether. Here, all the surfactants used in the present invention have a common property in that they are polyoxyalkylene-based nonionic surfactants.

The type of surfactant is not particularly limited.
Examples of polyoxyethylene distyrenated phenyl ethers include Emulgen A60 (“Emulgen” is a registered trademark; the same shall apply hereinafter), Emulgen A90 and Emulgen A500 manufactured by Kao Corporation.
For example, polyoxyethylene tribenzylphenyl ether includes Emulgen B66.
For example, polyoxyethylene lauryl ethers include Emulgen 103, Emulgen 104P, Emulgen 105, Emulgen 106, Emulgen 108, Emulgen 109P, Emulgen 120, Emulgen 123P, Emulgen 130K, Emulgen 147 or Emulgen 150.
For example, polyoxyethylene alkyl ethers include Emulgen 705, Emulgen 707, or Emulgen 709.
For example, polyoxyalkylene alkyl ethers include Emulgen LS106, Emulgen LS110, Emulgen LS114, or Emulgen MS110.
Examples of polyoxyethylene nonylphenyl ether include Nonion NS-210 (“Nonion” is a registered trademark. The same shall apply hereinafter).

Among them, the surfactant is at least one surfactant selected from the group consisting of Emulgen A60, Emulgen A90, Emulgen B66, Emulgen 108, Emulgen 120, Emulgen 707, Emulgen LS110, Emulgen LS114, and nonionic NS-210. is preferred.

The HLB of the surfactant used in the present invention is not limited as long as the effect of the present invention is achieved. A surfactant with an HLB of about 11 to 18 is preferred, and a surfactant with an HLB of about 12 to 16 is more preferred.

The concentration of the surfactant in the solution containing ASO is not particularly limited, but the upper limit of the range is preferably 20 g/L, more preferably 10 g/L, and the lower limit of the range is preferably 0.1 g/L. L, more preferably 0.5 g/L, is preferably used.

(Stabilization of ascorbate oxidase by combination of surfactant and buffer)
Another embodiment of the present invention provides ascorbate oxidase with polyoxyethylene distyrenated phenyl ether, polyoxyethylene tribenzylphenyl ether, polyoxyethylene lauryl ether, polyoxyethylene alkyl ether, polyoxyalkylene alkyl ether, and A method for stabilizing ascorbate oxidase, characterized by coexisting with a buffer solution containing at least one surfactant selected from the group consisting of polyoxyethylene nonylphenyl ether and an amine. By coexisting a specific polyoxyalkylene-based nonionic surfactant and a buffer solution containing an amine in this way, the actions of both components work together to synergistically exhibit a high stabilizing effect. This is clear from the results of Examples described later.

In the above method, when a surfactant and a buffer solution are combined, the type of surfactant is not particularly limited.
Examples of polyoxyethylene distyrenated phenyl ethers include Emulgen A60, Emulgen A90 and Emulgen A500 manufactured by Kao Corporation.
For example, polyoxyethylene tribenzylphenyl ether includes Emulgen B66.
For example, polyoxyethylene lauryl ethers include Emulgen 103, Emulgen 104P, Emulgen 105, Emulgen 106, Emulgen 108, Emulgen 109P, Emulgen 120, Emulgen 123P, Emulgen 130K, Emulgen 147, or Emulgen 150.
For example, polyoxyethylene alkyl ethers include Emulgen 705, Emulgen 707, or Emulgen 709.
For example, polyoxyalkylene alkyl ethers include Emulgen LS106, Emulgen LS110, Emulgen LS114, or Emulgen MS110.
For example, polyoxyethylene nonylphenyl ether includes nonion NS-210.

Among them, the surfactant is at least one surfactant selected from the group consisting of Emulgen A60, Emulgen A90, Emulgen B66, Emulgen 108, Emulgen 120, Emulgen 707, Emulgen LS110, Emulgen LS114, and nonionic NS-210. is preferred.

Furthermore, Emulgen 1108, Emulgen 1118S-70, Emulgen 1135S-70, Emulgen 1150S-60 (manufactured by Kao Corporation) and the like can be used as polyoxyethylene alkyl ethers other than those exemplified above.

In the method of the present invention, the amine-containing buffer to be used is not particularly limited. For example, N-(2-acetamido)-2-aminoethanesulfonic acid ( ACES)], N-(2-acetamido) iminodiacetic acid [N-(2-Acetamide) iminodiacetic acid (ADA)], N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid [N,N -Bis (2-hydroxyethyl)-2-aminoethanesulfonic acid (BES)], N, N-bis (2-hydroxyethyl) glycine [N, N-Bis (2-hydroxyethyl) glycine (Bicine)], bis (2- Hydroxyethyl) iminotris (hydroxymethyl) methane [Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris)], N-cyclohexyl-3-aminopropanesulfonic acid [N-Cyclohexyl-3-aminopropanesulfonic acid (CAPS) ], 3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid [3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid (EPPS)], 2-[4-( 2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid [2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES)], 2-hydroxy-3-[4-(2-hydroxyethyl )-1-piperazinyl]propanesulfonic acid [2-Hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPSO)], 2-morpholinoethanesulfonic acid, monohydrate [2- Morpholinoethanesulfonic acid, monohydrate (MES)], 3-morpholinopropanesulfonic acid [3-morpholinopropanesulfonic acid (MOPS)], 2-hydroxy-3-morpholinopropanesulfonic acid [2-Hydroxy-3-morpholinopropanesulfonic acid (MOPSO)], piperazine -1,4-bis(2-ethanesulfonic acid) [Piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES)], piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) , dihydrate [Piperazine-1,4-bis (2-hydroxy-3-propanesulfonic acid), dihydrate (POPSO)], N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid [N-Tris ( hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS)], N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid [N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES)], N-[Tris At least one selected from the group consisting of (hydroxymethyl)methyl]glycine [N-[Tris(hydroxymethyl)methyl]glycine (Tricine)] and tris(hydroxymethyl)aminomethane [Tris(hydroxymethyl)aminomethane (Tris)] Amines are mentioned.

In the method of the present invention, the amine-containing buffer contains N-(2-acetamido)iminodiacetic acid [N-(2-Acetamide)iminodiacetic acid (ADA)] and N,N-bis(2-hydroxyethyl)- It is preferably at least one selected from the group consisting of 2-aminoethanesulfonic acid [N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES)].

The combination of the buffer solution containing the amine and the surfactant used in the ASO stabilization method of the present invention is not particularly limited.

For example, when ADA is selected as a buffer solution containing an amine in the method, from the group consisting of Emulgen A60, Emulgen A90, Emulgen B66, Emulgen 108, Emulgen 120, Emulgen 707, Emulgen LS110, Emulgen LS114 and nonion NS-210 At least one selected surfactant is preferred.

Further, for example, when BES is selected as the buffer containing amine in the above method, it consists of Emulgen A60, Emulgen A90, Emulgen B66, Emulgen 108, Emulgen 120, Emulgen 707, Emulgen LS110, Emulgen LS114 and nonion NS-210. At least one surfactant selected from the group is preferred.

The concentration of the amine-containing buffer solution in the ASO-containing solution is not particularly limited, but the upper limit of the range is preferably 200 mM, more preferably 100 mM, and the lower limit of the range is preferably 5 mM, more preferably 20 mM. and should be used.

In addition, although the pH adjustment range of the buffer solution containing amine is not particularly limited, generally all enzymes and the like required for the measurement of biological samples exhibit stable properties near neutrality, and in the present invention, It is preferred to use a buffer. The upper limit of the range is preferably adjusted to 9.5, more preferably 7.5, and the lower limit of the range is preferably 4.5, more preferably 6.0.

ASO used in the present invention is an enzyme that catalyzes the following reactions classified under EC 1.10.3.3.
L-ascorbic acid + 1/2 O ₂ → dehydroascorbic acid + H ₂ O
Although the origin of the above enzyme is not particularly limited, it can be derived from, for example, those collected from plants such as cucumbers and pumpkins.
The ASO used in the present invention also includes enzymes that catalyze the following reactions described in JP-A-6-209770.
L-ascorbic acid + 1/2 O ₂ → dehydroascorbic acid + H ₂ O ₂
The origin of the enzyme is not particularly limited, but includes, for example, those collected from microorganisms. Examples of the microorganism include, but are not limited to, the genus Trichoderma, the genus Mortierella, and the genus Eupenicillium. In addition, the above enzyme may be chemically synthesized, or may be obtained by being expressed by genetic engineering. As long as it has ascorbate oxidase activity, it may be a mutant such as a protein in which a known ascorbate oxidase is mutated or a part of the domain is deleted.
The concentration of ASO in the solution containing ASO is not particularly limited, but varies depending on the origin of the enzyme, and is usually preferably used in the range of 0.1 to 50 U/mL.

In the method for stabilizing ASO of the present invention, a preservative or the like may be coexisted. Antiseptics are not particularly limited, but include, for example, azides, chelating agents, antibiotics, and antibacterial agents.

In addition, the ASO stabilization method of the present invention can be applied to various biochemical diagnostic methods. Examples of biochemical diagnostic methods include methods for measuring uric acid, free fatty acids, glucose, triglycerides, cholesterol, phospholipids, and the like.
Specifically, the ASO stabilization method of the present invention may coexist with other components necessary for the diagnostic method. For example, the components constituting the triglyceride measuring reagent generally include ASO, lipase, glycerol kinase, ATP, magnesium, peroxidase, chromogen, etc., and all of these may coexist, or A necessary one may be appropriately selected from among them and coexisted.

(Composition in which ascorbate oxidase is stabilized)
Another embodiment of the present invention is an ascorbate oxidase and an ascorbate oxidase-stabilized composition comprising (1) below.
(1) selected from the group consisting of polyoxyethylene distyrenated phenyl ether, polyoxyethylene tribenzylphenyl ether, polyoxyethylene lauryl ether, polyoxyethylene alkyl ether, polyoxyalkylene alkyl ether, and polyoxyethylene nonylphenyl ether; at least one surfactant.

The composition in which ascorbate oxidase is stabilized according to the present invention may be a composition containing ascorbate oxidase, the following (1) and the following (2).
(1) selected from the group consisting of polyoxyethylene distyrenated phenyl ether, polyoxyethylene tribenzylphenyl ether, polyoxyethylene lauryl ether, polyoxyethylene alkyl ether, polyoxyalkylene alkyl ether, and polyoxyethylene nonylphenyl ether; at least one surfactant,
(2) buffers containing amines;

The details of the buffer containing the surfactant and amine are as described in the section on the ASO stabilization method of the present invention.

The present invention may also be a biological component analysis kit containing any of the compositions described above.
Various biological component measurement methods have already been established in the technical field. Therefore, according to known methods, the ASO-stabilizing method and the ASO-containing composition of the present invention can be applied to biological component analysis kits to measure the amount or concentration of biological components in various samples. Its composition is not particularly limited.
The composition or kit of the present invention may be divided into two or more pieces in consideration of application to general-purpose automatic analyzers. In that case, it is preferable to allow the composition containing ASO to coexist with the surfactant and a buffer solution containing the amine that can be added as necessary. In addition, the compositions of the present invention (including the forms contained in kits) may be aqueous solutions or lyophilized. According to the present invention, it is possible to exhibit long-term storage stability even in liquid preparations in which ASO is likely to deactivate over time. Considering this point of view, the present invention can be used more effectively in liquid preparations (for example, liquid compositions such as aqueous solutions). In addition, even when a powdery (solid) preparation is prepared by freeze-drying or the like, for example, the deactivation of ASO after preparation in a liquid solvent can be suppressed and the stability can be enhanced. be.

The present invention also provides a method for producing a biological component analysis kit containing any of the compositions described above or any of the compositions described above.
In a method for producing a composition in which ascorbate oxidase is stabilized as described above, ascorbate oxidase and (1) polyoxyethylene distyrenated phenyl ether, polyoxyethylene tribenzyl phenyl ether, polyoxyethylene lauryl ether , polyoxyethylene alkyl ethers, polyoxyalkylene alkyl ethers, and polyoxyethylene nonylphenyl ethers. should be prepared. Further, in a method for producing a composition in which ascorbate oxidase is stabilized as described above, ascorbate oxidase and (1) polyoxyethylene distyrenated phenyl ether, polyoxyethylene tribenzylphenyl ether, polyoxyethylene At least one surfactant selected from the group consisting of lauryl ether, polyoxyethylene alkyl ether, polyoxyalkylene alkyl ether, and polyoxyethylene nonylphenyl ether, and (2) an amine-containing buffer solution coexist Alternatively, the compositions may be prepared according to any formulation technique known in the art. In these production methods, ascorbate oxidase, the polyoxyalkylene-based nonionic surfactant described in (1) above, and the amine described in (2) above, which is added as necessary, are included. The order of addition with the buffer solution is not particularly limited as long as the effects of the present invention are exhibited.
Here, the details of the polyoxyalkylene-based nonionic surfactant described in (1) above and the buffer solution containing the amine described in (2) above are described in detail below. As described in the Methods section.

As a further embodiment, the present invention provides (1) polyoxyethylene distyrenated phenyl ether, polyoxyethylene tribenzyl phenyl ether, polyoxyethylene tribenzyl phenyl ether, polyoxyethylene tribenzyl phenyl ether, polyoxyethylene distyrenated phenyl ether, polyoxyethylene tribenzyl phenyl ether, polyoxyethylene tribenzyl phenyl ether, polyoxyethylene Use of at least one surfactant selected from the group consisting of ethylene lauryl ether, polyoxyethylene alkyl ether, polyoxyalkylene alkyl ether, and polyoxyethylene nonylphenyl ether. Further, the present invention provides (1) polyoxyethylene distyrenated phenyl ether, polyoxyethylene tribenzyl phenyl ether, polyoxyethylene lauryl ether, for producing a composition containing stabilized ascorbate oxidase, Provided is the use of a buffer containing (2) an amine and at least one surfactant selected from the group consisting of polyoxyethylene alkyl ethers, polyoxyalkylene alkyl ethers, and polyoxyethylene nonylphenyl ethers. In the use in production of these, the order of addition of each component is not particularly limited as long as the effects of the present invention are exhibited.
Here, the details of the polyoxyalkylene-based nonionic surfactant described in (1) above and the buffer solution containing the amine described in (2) above are described in detail below. As described in the Methods section.

As a further embodiment, the present invention provides (1) polyoxyethylene distyrenated phenyl ether, polyoxyethylene tribenzyl phenyl ether, polyoxyethylene lauryl ether, polyoxyethylene for stabilizing ascorbic acid oxidase. Also provided are compositions comprising at least one surfactant selected from the group consisting of ethylene alkyl ethers, polyoxyalkylene alkyl ethers, and polyoxyethylene nonylphenyl ethers. Furthermore, the present invention provides (1) polyoxyethylene distyrenated phenyl ether, polyoxyethylene tribenzylphenyl ether, polyoxyethylene lauryl ether, polyoxyethylene alkyl ether, polyoxyethylene for stabilizing ascorbic acid oxidase. Provided is a composition comprising at least one surfactant selected from the group consisting of alkylene alkyl ethers and polyoxyethylene nonylphenyl ethers, and (2) a buffer solution containing an amine. These compositions contain the polyoxyalkylene-based nonionic surfactant described in (1) above, and, if necessary, a buffer solution containing the amine described in (2) above. Other optional components such as additives may be included as long as the effects of the above are not impaired, and the order of addition of each component when producing these compositions does not matter.
Here, the details of the polyoxyalkylene-based nonionic surfactant described in (1) above and the buffer solution containing the amine described in (2) above are described in the method for stabilizing ASO of the present invention. as explained in Section 1.

(Ascorbate oxidase activity measurement method)
ASO activity measurement is performed under the following measurement conditions.
<Reaction solution>
100 mM phosphate buffer pH 5.6
0.5 mM L-ascorbic acid <measurement conditions>
(1) The above reaction solution is placed in a 1 mM cuvette and preheated at 30° C. for about 5 minutes.
(2) Add 0.1 mL of the enzyme solution to initiate the reaction. After reacting for exactly 5 minutes, 3.0 mL of 0.2N hydrochloric acid solution is added to stop the reaction. The absorbance of this solution at 245 nm is measured with a spectrophotometer.
(3) A blank test is prepared by leaving the reaction solution at 30° C. for 5 minutes, adding 3.0 mL of 0.2N hydrochloric acid solution and mixing, and then adding 0.1 mL of the enzyme solution. The steepness of this liquid is similarly measured.

In the present invention, "ascorbate oxidase is stable" means that deactivation of ascorbate oxidase over time is suppressed and long-term storage stability is improved. Specifically, after storage at 35°C for 7 days, the It means that the residual enzymatic activity of ASO is enhanced.

EXAMPLES Hereinafter, the present invention will be specifically described with reference to Examples. In addition, this invention is not specifically limited by an Example.

Example 1: Stabilization of ascorbic acid oxidase by surfactant ASO (Toyobo ASO-311) 3U/mL was dissolved in distilled water containing magnesium chloride (1g/L), and various additives were added to this aqueous solution at 1g/mL. The mixture was stored at 37° C. for 7 days and examined for residual activity (ratio of activity value after storage to [start activity] immediately after dissolution).

Table 1 shows the results.
By adding Emulgen A60, Emulgen A90, Emulgen B66, Emulgen 108, Emulgen 120, Emulgen 707, Emulgen LS110, Emulgen LS114, or Nonion NS-210 to the ASO aqueous solution, good stability is obtained compared to the case where no additive is added. Got.

Example 2: Stabilization of Ascorbic Acid Oxidase by Combination of Surfactant and Buffer 3 U/mL of ASO (ASO-311 manufactured by Toyobo) in distilled water containing magnesium chloride (1 g/L), ADA (100 mM, pH 7.0). 0), or dissolved in BES (100 mM, pH 7.0), added 1 g / L of various additives to this aqueous solution, and stored at 37 ° C. for 7 days as a control when no additives were added to distilled water. The activity (the activity value immediately after dissolution = the ratio of the activity value after storage to [start activity]) was examined.
For reference, data was also obtained on the stabilization of ascorbate oxidase in a buffer solution alone, and good stability against distilled water was obtained by dissolving ASO in ADA or BES.

The results are shown in Tables 2 and 3.
Synergistic effects of amine-containing buffers (ADA or BES) and various additives were investigated. Synergy was determined as follows.
[Stabilization effect overall (A)]
The result of the comparative example was subtracted from the experimental result of each example, and the stabilizing effect was made relative.
(Activity residual rate % of each example)-(Activity residual rate (12%) in comparative example (distilled water × no additive))
[Stabilizing effect Buffer (B)]
The stabilizing effect of each buffer (ADA or BES) alone was determined.
(Residual activity rate % without additive) - (Residual activity rate (12%) in comparative example (distilled water x no additive))
[Stabilizing effect additive (C)]
The stabilizing effect of each additive alone was determined.
(Residual activity rate % without buffer) - (Residual activity rate (12%) in comparative example (distilled water x no additive))
[Synergy]
Overall (A) - Buffer (B) - Additive (C)
No synergy should be 0%. Therefore, it was determined that a positive value indicates a positive synergistic effect, and a negative value indicates a negative synergistic effect.

According to the results in Table 2, the ADA buffer and at least one selected from the group consisting of Emulgen A60, Emulgen A90, Emulgen B66, Emulgen 108, Emulgen 120, Emulgen 707, Emulgen LS110, Emulgen LS114, and nonion NS210 A synergistic effect in the stabilizing effect was observed in combination with the additives.

According to the results of Table 3, the BES buffer and at least one selected from the group consisting of Emulgen A60, Emulgen A90, Emulgen B66, Emulgen 108, Emulgen 120, Emulgen 707, Emulgen LS110, Emulgen LS114, and nonion NS210 A synergistic effect in the stabilizing effect was observed in combination with the additives.

Since ascorbate oxidase in solution is very unstable, the improvement in storage stability achieved by the present invention, as shown in the results of the examples under the test conditions described above, is highly significant. Beneficial. In particular, analytical reagents for molecular biology using the functions of enzymes, analytical reagents for biochemistry, in-vitro diagnostics, liquid in-vitro diagnostics, dry in-vitro diagnostics processed into chips or slits, enzyme sensors Enzymatic reaction is the most important factor and can be the rate-limiting factor in compositions such as , enzyme electrodes, pharmaceuticals, foods and beverages. Therefore, as shown in the results of the above Examples, it is extremely remarkable that the storage stability of ascorbate oxidase can be enhanced in a solution that tends to be destabilized, and that the stability can be enhanced by a very simple technique. proved to be beneficial.

It should be noted that the embodiments and examples disclosed above are all illustrative and not restrictive. Further, embodiments and examples in which the contents disclosed in the embodiments and examples are combined are also included in the scope of the present invention. The technical scope of the present invention is valid according to the scope of claims, and includes all changes, modifications, replacements, etc. within the meaning and range of equivalents to the description of the scope of claims.

INDUSTRIAL APPLICABILITY According to the present invention, a liquid reagent containing ascorbate oxidase (ASO) that is stable for a longer period of time can be provided.

Claims (15)

ascorbic acid oxidase in the group consisting of polyoxyethylene distyrenated phenyl ether, polyoxyethylene tribenzyl phenyl ether, polyoxyethylene lauryl ether, polyoxyethylene alkyl ether, polyoxyalkylene alkyl ether, and polyoxyethylene nonylphenyl ether; At least one surfactant selected from the group consisting of N-(2-acetamido)iminodiacetic acid (ADA) and N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) A method for stabilizing ascorbate oxidase, which comprises coexisting with a buffer containing at least one selected amine . The method for stabilizing ascorbate oxidase according to claim 1 , wherein the amine-containing buffer solution is N 1 ,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES 3 ) . The method for stabilizing ascorbate oxidase according to claim 1 or 2, wherein the ascorbate oxidase is stable even when stored at 35°C for 7 days. The stabilization method according to any one of claims 1 to 3, wherein ascorbate oxidase is stabilized in a liquid composition. 5. The stabilization method according to claim 4, wherein ascorbate oxidase is allowed to coexist in the liquid composition at a concentration of 0.1 to 50 U/mL. 6. The stabilization method according to claim 4, wherein the surfactant is coexistent in the liquid composition at a concentration of 0.1 to 20 g/L. The stabilization method according to any one of claims 4 to 6, wherein the buffer solution containing the amine is allowed to coexist in the liquid composition at a concentration of 5 to 200 mM. An ascorbate oxidase-stabilized composition comprising ascorbate oxidase, (1) below and (2) below.
(1) selected from the group consisting of polyoxyethylene distyrenated phenyl ether, polyoxyethylene tribenzylphenyl ether, polyoxyethylene lauryl ether, polyoxyethylene alkyl ether, polyoxyalkylene alkyl ether, and polyoxyethylene nonylphenyl ether; at least one surfactant,
(2) at least one amine selected from the group consisting of N-(2-acetamido)iminodiacetic acid (ADA) and N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES); buffer containing. 9. The composition of claim 8, wherein the amine-containing buffer is N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES). 10. A composition according to claim 8 or 9, which is stable when stored at 35[deg.]C for 7 days. The composition according to any one of claims 8 to 10, which is a liquid composition. The composition according to claim 11, wherein the concentration of ascorbate oxidase in the liquid composition is 0.1-50 U/mL. The composition according to claim 11 or 12, wherein the concentration of said surfactant in the liquid composition is 0.1-20 g/L. The composition according to any one of claims 11 to 13, wherein the concentration of said amine-containing buffer in the liquid composition is 5 to 200 mM. A biological component analysis kit comprising the composition according to any one of claims 8 to 14 .