Method for stabilizing and preserving fructosyl peptide oxidase in the presence of methylene blue or chromogen producing methylene blue, and aqueous solution

Abstract

Disclosed is a method for stabilizing a fructosyl peptide oxidase in the presence of methylene blue or a chromogen that produces methylene blue, the method comprising a step for preparing an aqueous solution containing a fructosyl peptide oxidase, methylene blue or a chromogen that produces methylene blue, and at least one compound selected from the group consisting of sodium azide and tryptophan.

Description

Method for stabilizing and preserving fructosylpeptide oxidase in the presence of methylene blue or a chromogen that produces methylene blue, and aqueous solution

[0001] The present invention relates to a method for stabilizing fructosylpeptide oxidase in the presence of methylene blue or a chromogen that produces methylene blue.

[0002] Glycated proteins are contained in body fluids such as blood and biological samples such as hair in vivo. The concentration of glycated proteins present in blood depends on the concentration of sugars such as glucose dissolved in serum. In the field of clinical diagnosis, the measurement of the concentration of hemoglobin A1c (hereinafter referred to as HbA1c), which is a glycated protein in blood, is used for the diagnosis and monitoring of diabetes (see Non-Patent Document 1). Hemoglobin is a hemoprotein with a molecular weight of 64,000, having two subunits each of α-chain and β-chain. HbA1c is particularly defined as hemoglobin in which the N-terminal valine residue of the β-chain is glycated. As methods for measuring this HbA1c, instrumental analysis methods using high performance liquid chromatography (HPLC) (see Non-Patent Document 2), immunoassay methods using antigen-antibody reactions (Non-Patent Document 3), etc. have been known.

[0003] In recent years, the development of enzymatic methods for the measurement of HbA1c that can be applied to general-purpose automated analyzers and are easy to operate has progressed, and various methods have been reported. The enzymatic methods for the measurement of HbA1c reported so far mainly use proteases and glycated peptide oxidases. One known method using proteases and glycated peptide oxidases involves reacting HbA1c in a sample with a protease to produce a glycated peptide called fructosyl dipeptide (Fru-Val-His), reacting the produced fructosyl dipeptide with fructosyl peptide oxidase to produce hydrogen peroxide, and measuring the produced hydrogen peroxide to measure the HbA1c in the sample (see Patent Document 1). Furthermore, HbA1c measurement kits are sold by several manufacturers. For example, "Metabolid HbA1c" (Non-Patent Literature 4) is a known HbA1c measurement kit consisting of a first reagent containing a phenothiazine-based leuco-type chromopropyl alcohol that reacts with hydrogen peroxide to produce color, and a second reagent containing fructosyl peptide oxidase. In HbA1c measurement kits, leuco-type chromopropyl alcohol is often used to perform measurements with high sensitivity.

[0004] In the enzymatic assay of HbA1c, it is desirable that fructosyl peptide oxidase (fructosyl peptide oxidase), used in the HbA1c assay, remain stable and do not become inactive during storage of the HbA1c assay reagent. A method for stabilizing fructosyl peptide oxidase is known, which involves coexisting a fructosyl peptide oxidase-containing solution with a polyaminocarboxylic acid-based chelating reagent and one or more reagents selected from ammonium salts, oxycarboxylic acid-based chelating reagents, sugar alcohols, and amino acids (see Patent Document 2).

[0005] Japanese Patent Publication No. 2001-095598 Japanese Patent Publication No. 2006-325547

[0006] Clin Chem Lab Med, Vol. 36, pp. 299-308 (1998). Diabetes, Vol. 27, No. 2, pp. 102-107 (1978). Journal of the Japanese Society for Clinical Laboratory Automation, Vol. 18, No. 4, p. 620 (1993). Package insert for the glycated hemoglobin A1c kit "Metabolead HbA1c", revised April 2024 (9th edition).

[0007] The present inventors prepared an HbA1c measurement kit containing a reagent in which fructosyl peptide oxidase and a leuco-type chromogen coexist in an aqueous medium. They found that when fructosyl peptide oxidase was coexisted in an aqueous medium with a leuco-type chromogen that produces methylene blue or with methylene blue itself, the stability of the fructosyl peptide oxidase decreased. Therefore, the object of the present invention is to provide a stabilization method and a storage method for stably storing fructosyl peptide oxidase in an aqueous medium even in the presence of methylene blue or a methylene blue-producing chromogen, and to provide an aqueous solution in which fructosyl peptide oxidase is stabilized even in the presence of methylene blue or a methylene blue-producing chromogen.

[0008] As a result of diligent research, the inventors of the present invention have found that fructosyl peptide oxidase can be stabilized by adding at least one compound selected from the group consisting of sodium azide and tryptophan to an aqueous solution containing methylene blue or a chromogen that produces methylene blue and fructosyl peptide oxidase, thereby completing the present invention.

[0009] The present invention includes the following embodiments: 1. A method for stabilizing fructosyl peptide oxidase in the presence of methylene blue or a methylene blue-producing chromogen, comprising the step of preparing an aqueous solution containing fructosyl peptide oxidase, methylene blue or a methylene blue-producing chromogen, and at least one compound selected from the group consisting of sodium azide and tryptophan. 2. The method according to claim 1, further comprising the step of storing the aqueous solution. 3. A method for storing methylene blue or a methylene blue-producing chromogen, comprising the step of storing an aqueous solution containing fructosyl peptide oxidase, methylene blue or a methylene blue-producing chromogen, and at least one compound selected from the group consisting of sodium azide and tryptophan. 4. The method according to any one of claims 1 to 3, wherein the methylene blue-producing chromogen is a phenothiazine-based chromogen. 5. The method according to claim 4, wherein the phenothiazine-based chromogen is 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt. Claim 6. The method according to any one of claims 1 to 5, wherein the concentration of at least one compound selected from the group consisting of sodium azide and tryptophan in the aqueous solution is 0.01 g / L to 20 g / L. Claim 7. The method according to any one of claims 1 to 6, wherein the concentration of methylene blue in the aqueous solution is 0.01 mg / L to 10 mg / L, or the concentration of the chromogen that produces methylene blue in the aqueous solution is 1 mg / L to 50 mg / L. Claim 8. The method according to any one of claims 1 to 7, wherein the aqueous solution is contained in a container with a light-shielding rate of 70% or less. Claim 9. The method according to claim 2 or 3, wherein the storage period in the step of storing the aqueous solution is 1 day to 2 years. Claim 10. The method according to any one of claims 1 to 9, wherein the aqueous solution does not contain a proteolytic enzyme. Claim 11. An aqueous solution containing a fructosyl peptide oxidase, methylene blue or a chromogen that produces methylene blue, and at least one compound selected from the group consisting of sodium azide and tryptophan.Item 12. The aqueous solution according to Item 11, wherein the chromogen that produces methylene blue is a phenothiazine-based chromogen. Item 13. The aqueous solution according to Item 12, wherein the phenothiazine-based chromogen is 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt. Item 14. The aqueous solution according to any one of Items 11 to 13, wherein the concentration of at least one compound selected from the group consisting of sodium azide and tryptophan is 0.01 g / L to 20 g / L. Item 15. The aqueous solution according to any one of Items 11 to 14, wherein the concentration of methylene blue is 0.01 mg / L to 10 mg / L, or the concentration of the chromogen that produces methylene blue is 1 mg / L to 50 mg / L. Item 16. The aqueous solution according to any one of Items 11 to 15, contained in a container with a light-shielding rate of 70% or less. Item 17. An aqueous solution according to any one of claims 11 to 16, which does not contain a proteolytic enzyme.

[0010] According to the present invention, a stabilization method and storage method are provided for stably storing fructosyl peptide oxidase in an aqueous medium, even in the presence of methylene blue or a chromogen that produces methylene blue, and an aqueous solution is provided in which fructosyl peptide oxidase is stabilized even in the presence of methylene blue or a chromogen that produces methylene blue.

[0011] A method for stabilizing fructosyl peptide oxidase in the presence of methylene blue or a methylene blue-producing chromogen, according to one aspect of the present invention, includes the step of preparing an aqueous solution containing fructosyl peptide oxidase, methylene blue or a methylene blue-producing chromogen, and at least one compound selected from the group consisting of sodium azide and tryptophan. In the aqueous solution prepared in this step, fructosyl peptide oxidase is stabilized by sodium azide and / or tryptophan, even in the presence of methylene blue or a methylene blue-producing chromogen, which has the effect of destabilizing fructosyl peptide oxidase. In this specification, stabilization of fructosyl peptide oxidase means that the decrease in the enzyme activity of fructosyl peptide oxidase is suppressed.

[0012] Fructosyl peptide oxidase is an oxidase that, in the presence of oxygen, uses fructosyl peptides as substrates to produce glucosone, peptides, and hydrogen peroxide. The origin of fructosyl peptide oxidase is not particularly limited; for example, it may be wild-type fructosyl peptide oxidase derived from microorganisms such as fungi and bacteria, or a recombinant thereof.

[0013] The chromophores that produce methylene blue are not particularly limited as long as they are compounds that produce methylene blue by oxidation, and may be, for example, phenothiazine chromophores. Phenothiazine chromophores are chromophores that have a phenothiazine skeleton. Examples of phenothiazine chromophores include 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine, 10-N-(acetylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine, 10-N-(phenylcarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine, 10-N-(3-(methylcarboxyamino)-hexamethyl-amino)-3,7-bis(dimethylamino)-10H-phenothiazine, and 10-N-(((3-(methylcarboxyamino)-4-methyl)-phenyl)-amino)-3,7 Examples include -bis(dimethylamino)-10H-phenothiazine, 10-N-((3-(methylcarboxyaminomethyl)-phenyl)-methylamino)-3,7-bis(dimethylamino)phenothiazine, 10-N-(1-naphthaleneamino)-3,7-bis(dimethylamino)-10H-phenothiazine, 10-N-(methyl)-3,7-bis(dimethylamino)-10H-phenothiazine, 10-N-(phenylamino)-3,7-bis(dimethylamino)-10H-phenothiazine, 10-N-(methylamino)-3,7-bis(dimethylamino)-10H-phenothiazine, or salts thereof. The phenothiazine chromogen may be 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine or a salt thereof, or 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt.

[0014] The above aqueous solution may contain other components such as buffers, pH adjusters, chelating agents, peroxidases, and surfactants, as long as they do not interfere with the stabilization of fructosyl peptide oxidase and the storage of the aqueous solution. Preferably, the aqueous solution does not contain proteolytic enzymes or fructosyl peptides.

[0015] Examples of buffering agents include lactic acid buffers, citrate buffers, acetate buffers, succinate buffers, phthalate buffers, phosphate buffers, triethanolamine buffers, diethanolamine buffers, lysine buffers, barbiturate buffers, imidazole buffers, malic acid buffers, oxalate buffers, glycine buffers, borate buffers, carbonate buffers, Tris buffers, and Good's buffers. Examples of Good's buffers include 2-morpholinoethanesulfonic acid (MES), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), N-(2-acetamide)iminodiacetic acid (ADA), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-[N-(2-acetamide)amino]ethanesulfonic acid (ACES), and 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO). ), 2-[N,N-bis(2-hydroxyethyl)amino]ethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), 2-{N-[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), N-(2-hydroxyethyl)-N'-(2-sulfoethyl)piperazine (HEPES), 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), 2 -Hydroxy-3-{[N-Tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPSO), piperazine-N,N'-bis(2-hydroxypropane-3-sulfonic acid) (POPSO), N-(2-hydroxyethyl)-N'-(2-hydroxy-3-sulfopropyl)piperazine (HEPPSO), N-(2-hydroxyethyl)-N'-(3-sulfopropyl)piperazine (EPPS), N-Tris(hydroxymethyl) Examples include methylglycine (tricine), N,N-bis(2-hydroxyethyl)glycine (bicine), 3-[N-tris(hydroxymethyl)methyl]aminopropanesulfonic acid (TAPS), 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), 3-(N-cyclohexylamino)-2-hydroxypropanesulfonic acid (CAPSO), and 3-(N-cyclohexylamino)propanesulfonic acid (CAPS).

[0016] The pH of the above aqueous solution may be, for example, 4 to 11, 5 to 10, 6 to 9, or 7 to 8.

[0017] The concentration of fructosyl peptide oxidase in the aqueous solution may be, for example, 0.01 kU / L or higher, 0.05 kU / L or higher, 0.1 kU / L or higher, 0.5 kU / L or higher, 1.0 kU / L or higher, 1.5 kU / L or higher, or 2.0 kU / L or higher. Alternatively, the concentration of fructosyl peptide oxidase in the aqueous solution may be, for example, 50 kU / L or less, 40 kU / L or less, 30 kU / L or less, 25 kU / L or less, 20 kU / L or less, 15 kU / L or less, 12 kU / L or less, 10 kU / L or less, 7 kU / L or less, or 5 kU / L or less. The above values ​​can be freely combined. For example, the concentration of fructosyl peptide oxidase in the aqueous solution may be 0.01 kU / L to 30 kU / L, 0.1 to 15 kU / L, 0.5 to 12 kU / L, or 1 to 10 kU / L.

[0018] The concentration of methylene blue in the aqueous solution may be, for example, 0.01 mg / L or more, 0.05 mg / L or more, 0.1 mg / L or more, 0.2 mg / L or more, 0.3 mg / L or more, 0.4 mg / L or more, 0.5 mg / L or more, 0.6 mg / L or more, 0.7 mg / L or more, 0.8 mg / L or more, 0.9 mg / L or more, 1 mg / L or more, 2 mg / L or more, 3 mg / L or more, or 4 mg / L or more. Alternatively, the concentration of methylene blue in the aqueous solution may be, for example, 10 mg / L or less, 9 mg / L or less, 8 mg / L or less, 7 mg / L or less, 6 mg / L or less, or 5 mg / L or less. The above values ​​can be freely combined. For example, the concentration of methylene blue in the aqueous solution may be 0.01 mg / L to 10 mg / L, 0.1 mg / L to 7 mg / L, or 1 mg / L to 5 mg / L.

[0019] The concentration of the chromogen that produces methylene blue in the above aqueous solution may be, for example, 0.1 mg / L or more, 0.5 mg / L or more, 1 mg / L or more, 2 mg / L or more, 3 mg / L or more, 4 mg / L or more, 5 mg / L or more, 6 mg / L or more, 7 mg / L or more, 8 mg / L or more, 9 mg / L or more, 10 mg / L or more, 15 mg / L or more, or 20 mg / L or more. Alternatively, the concentration of the chromogen that produces methylene blue in the above aqueous solution may be, for example, 200 mg / L or less, 150 mg / L or less, 100 mg / L or less, 50 mg / L or less, 40 mg / L or less, 30 mg / L or less, or 25 mg / L or less. The above values ​​can be freely combined. For example, the concentration of the colorant that produces methylene blue in the aqueous solution may be 1 mg / L to 50 mg / L, 5 mg / L to 40 mg / L, or 15 mg / L to 30 mg / L.

[0020] The concentration of at least one compound selected from the group consisting of sodium azide and tryptophan in the above aqueous solution may be, for example, 0.01 g / L or more, 0.05 g / L or more, 0.1 g / L or more, 0.2 g / L or more, 0.3 g / L or more, 0.4 g / L or more, 0.5 g / L or more, 1 g / L or more, 2 g / L or more, or 3 g / L or more. The concentration of at least one compound selected from the group consisting of sodium azide and tryptophan in the above aqueous solution may be, for example, 30 g / L or less, 20 g / L or less, 10 g / L or less, 9 g / L or less, 8 g / L or less, 7 g / L or less, 6 g / L or less, or 5 g / L or less. The above values ​​can be freely combined. For example, the concentration of at least one compound selected from the group consisting of sodium azide and tryptophan in the aqueous solution may be 0.01 g / L to 20 g / L, 0.05 g / L to 10 g / L, or 0.1 g / L to 5 g / L.

[0021] The concentration of the buffering agent in the aqueous solution may be, for example, 0.1 g / L to 50 g / L, 0.5 g / L to 30 g / L, or 1 g / L to 20 g / L.

[0022] The container for containing the above aqueous solution is not particularly limited and may be, for example, a transparent or translucent container. A "transparent or translucent container" is a container that transmits light (more specifically, visible light) at least partially. The light-shielding rate of the container may be, for example, 99% or less, 98% or less, 95% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less. The light-shielding rate of the container may be, for example, 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more. The light-shielding rate of the container can be determined by, for example, the same method as in JIS L 1055:2009, but it may also be determined by a simpler method. For example, the illuminance meter is positioned so that the light hitting the outer surface of the container hits the light receiving part, and the illuminance L ０ (lx) is measured, and the illuminance meter is placed inside the container to measure the illuminance L ｔ The illuminance (lx) may be measured, and the light shielding rate may be determined from these measured values ​​using the following formula. Alternatively, for example, the illuminance meter may be positioned so that the light hitting the outer surface of the container hits the light receiving part, and the illuminance L may be measured. ０ (lx) is measured, a portion of the wall is cut out from the container, and the test piece is placed on the illuminance meter so that only the light that passes through the cut-out test piece reaches the light receiving part of the illuminance meter, and the illuminance L is measured. ｔ (lx) may be measured, and the light shielding rate may be determined from these measured values ​​using the following formula (the light shielding rate in the example was measured using this method). Light shielding rate (%) = (1 - L ｔ / L ０ ) × 100 According to the inventors' findings, the stability of fructosyl peptide oxidase in the presence of methylene blue or a chromogen that produces methylene blue tends to decrease particularly when exposed to light. However, in the above aqueous solution, fructosyl peptide oxidase is stabilized by sodium azide and / or tryptophan even under light irradiation. Therefore, not only light-shielding containers but also transparent or translucent containers as described above can be used as containers for the aqueous solution.

[0023] The above aqueous solution can be prepared by adding fructosyl peptide oxidase, methylene blue or a chromogen that produces methylene blue, at least one compound selected from the group consisting of sodium azide and tryptophan, and optionally other components to an aqueous medium. The order in which these components are added is not particularly limited. For example, fructosyl peptide oxidase and methylene blue or a chromogen that produces methylene blue may be added to an aqueous medium, and sodium azide and / or tryptophan may be added to the resulting aqueous solution. Alternatively, fructosyl peptide oxidase and sodium azide and / or tryptophan may be added to an aqueous medium, and methylene blue or a chromogen that produces methylene blue may be added to the resulting aqueous solution. The aqueous medium may be water, or a mixed solvent of water and a water-soluble solvent, and the water may be deionized water or distilled water.

[0024] This stabilization method may further include a step of storing the aqueous solution. In this specification, "storing" the aqueous solution means keeping the aqueous solution in a container for a predetermined period (typically several hours to several years) with the aim of maintaining the state of the aqueous solution (composition, chemical properties, physical properties, etc.) at the start of storage. Therefore, if the state of the aqueous solution changes unintentionally due to factors such as temperature, humidity, light, and the stability of the components while the aqueous solution is being stored in the container, such storage is included in the scope of "storage". On the other hand, if the components of the aqueous solution are intentionally reacted with each other while the aqueous solution is being stored in the container, such storage is not included in the scope of "storage". For example, if an aqueous solution containing fructosyl peptide oxidase, methylene blue or a chromogen that produces methylene blue, and sodium azide and / or tryptophan, plus other components that react with these components (e.g., a substrate for fructosyl peptide oxidase), is kept in a container for a predetermined period to allow the reaction to proceed, this is not considered "storage". Furthermore, if an aqueous solution is kept in a container for a very short period (typically a few seconds to a few minutes) for the purpose of measuring or preparing for various measurements such as fluorescence intensity, this is not considered "storage."

[0025] The storage period for the above aqueous solution is not particularly limited and may be, for example, 1 hour to 2 years, 1 day to 2 years, 1 day to 1 year, 1 day to 6 months, 1 day to 1 month, or 1 day to 23 days. The storage temperature is not particularly limited and may be, for example, 1°C to 50°C, 2°C to 30°C, 2°C to 16°C, 2°C to 8°C, or 4°C.

[0026] As described above, according to the present invention, fructosyl peptide oxidase can be stably stored even in the presence of methylene blue or a methylene blue-producing chromogen. Therefore, another aspect of the present invention is a method for storing fructosyl peptide oxidase in the presence of methylene blue or a methylene blue-producing chromogen, comprising the step of storing an aqueous solution (i.e., the above-mentioned aqueous solution) containing fructosyl peptide oxidase, methylene blue or a methylene blue-producing chromogen, and at least one compound selected from the group consisting of sodium azide and tryptophan. Details of the aqueous solution containing fructosyl peptide oxidase, methylene blue or a methylene blue-producing chromogen, and at least one compound selected from the group consisting of sodium azide and tryptophan, the container for containing the aqueous solution, the storage period, and the storage temperature are the same as those for the stabilization method described above.

[0027] Another aspect of the present invention is the aqueous solution itself, which contains fructosyl peptide oxidase, methylene blue or a chromogen that produces methylene blue, and at least one compound selected from the group consisting of sodium azide and tryptophan. As described above, in this aqueous solution, fructosyl peptide oxidase is stabilized by sodium azide and / or tryptophan, even in the presence of methylene blue or a chromogen that produces methylene blue, which has the effect of destabilizing fructosyl peptide oxidase.

[0028] This aqueous solution may be contained in a container. The details of the container are the same as those for the stabilization method described above.

[0029] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.

[0030] In the following examples, reagents from the following manufacturers were used: ADA (N-(2-acetamide)iminodiacetic acid; manufactured by Dojin Chemical Laboratories Co., Ltd.), DA-67 (10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt; manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), dimethyl sulfoxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), sodium azide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), FPOX-CET (fructosyl peptide oxidase; manufactured by Kikkoman Corporation), sodium hydroxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), tryptophan (L-tryptophan; manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). Histidine (L-histidine; manufactured by Nacalai Tesque), disodium hydrogen phosphate (manufactured by Fujifilm Wako Pure Chemical Industries), sodium dihydrogen phosphate (manufactured by Fujifilm Wako Pure Chemical Industries), EMSE (N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine; manufactured by Daito Chemix), 4-AA (4-aminoantipyrine; manufactured by Actec), peroxidase (manufactured by Toyobo), 1-deoxyfructosylvaline (manufactured by Peptide Laboratories), physiological saline (Otsuka Saline Injection; manufactured by Otsuka Pharmaceutical Factory Co., Ltd.), and methylene blue (manufactured by Nacalai Tesque) were used.

[0031] [Example 1] Preparation of an aqueous solution containing fructosyl peptide oxidase The following components were added to water to prepare an enzyme-containing aqueous solution A containing FPOX-CET (fructosyl peptide oxidase). The following concentrations of each component are the concentrations in the enzyme-containing aqueous solution A. ADA 5.7 g / L DA-67 26.0 mg / L Dimethyl sulfoxide 1.032 g / L Sodium azide 0.3 g / L FPOX-CET 2 kU / L The pH was adjusted to 7.0 with an appropriate amount of sodium hydroxide. Dimethyl sulfoxide is a solvent for dissolving DA-67, and the enzyme-containing aqueous solution A was prepared using a solution in which DA-67 was dissolved in dimethyl sulfoxide.

[0032] [Example 2] Preparation of an aqueous solution containing fructosyl peptide oxidase An aqueous solution B containing enzyme was prepared in the same manner as in Example 1's aqueous solution A, except that "0.3 g / L sodium azide" in the enzyme-containing aqueous solution A was changed to "3 g / L tryptophan".

[0033] [Comparative Example 1] Preparation of an aqueous solution containing fructosyl peptide oxidase An aqueous solution a containing enzyme was prepared in the same manner as in Example 1, except that "0.3 g / L sodium azide" was not added to the aqueous solution A containing enzyme.

[0034] [Comparative Example 2] Preparation of an aqueous solution containing fructosyl peptide oxidase An aqueous solution b containing enzyme was prepared in the same manner as in Example 1's aqueous solution A, except that the "sodium azide 0.3 g / L" in the enzyme-containing aqueous solution A was changed to "histidine 3 g / L".

[0035] [Example 3] Measurement of the activity of fructosyl peptide oxidase in each aqueous solution Using an automated analyzer Hitachi 7170 (manufactured by Hitachi High-Tech Corporation) as the measuring instrument, the activity of fructosyl peptide oxidase in enzyme-containing aqueous solution A of Example 1 and enzyme-containing aqueous solution B of Example 2 was measured using the following activity measurement kit and procedure.

[0036] <Activity Measurement Kit> (Reagent 1) 100 mmol / L Phosphate buffer (pH 8.0) (Prepared using disodium hydrogen phosphate and sodium dihydrogen phosphate) EMSE 0.3 g / L 4-AA 0.1 g / L Peroxidase 3 kU / L (Reagent 2) 100 mmol / L Phosphate buffer (pH 8.0) (Prepared using disodium hydrogen phosphate and sodium dihydrogen phosphate) 1-Deoxyfructosylvaline 1.7 mmol / L

[0037] <Activity Measurement Procedure> An enzyme-containing aqueous solution (2.0 μL) and the first reagent (150 μL) of the above-mentioned activity measurement kit were added to a reaction cell and allowed to stand at 37°C for 5 minutes. The absorbance (E1) of the mixture after standing was measured at a primary wavelength of 546 nm and a secondary wavelength of 700 nm. Next, the second reagent (50 μL) of the above-mentioned activity measurement kit was added to this mixture and allowed to react at 37°C for 5 minutes. The absorbance (E2) of the reaction solution was measured at a primary wavelength of 546 nm and a secondary wavelength of 700 nm. The absorbance difference ΔE was calculated by subtracting E1 from E2 and was taken as the absorbance of the enzyme-containing aqueous solution. The absorbance difference ΔE' was calculated in the same manner, except that physiological saline was used instead of the enzyme-containing aqueous solution, and was taken as the absorbance of physiological saline. The value obtained by subtracting the absorbance difference ΔE' of physiological saline from the absorbance difference ΔE of the enzyme-containing aqueous solution was taken as the enzyme activity value of the enzyme-containing aqueous solution.

[0038] [Comparative Example 3] Measurement of fructosyl peptide oxidase activity in each aqueous solution The enzyme activity values ​​for enzyme-containing aqueous solution a of Comparative Example 1 and enzyme-containing aqueous solution b of Comparative Example 2 were calculated in the same manner as in Example 3, except that enzyme-containing aqueous solution a of Comparative Example 1 or enzyme-containing aqueous solution b of Comparative Example 2 were used instead of enzyme-containing aqueous solution A of Example 1 or enzyme-containing aqueous solution B of Example 2.

[0039] [Example 4] Evaluation of residual activity of fructosyl peptide oxidase in each aqueous solution after storage The residual activity of fructosyl peptide oxidase after storage was evaluated for each of the enzyme-containing aqueous solution A of Example 1 and enzyme-containing aqueous solution B of Example 2 according to the following procedure.

[0040] (1) Storage of enzyme-containing aqueous solution: The enzyme-containing aqueous solution was filled into a transparent polystyrene container or a brown translucent polyethylene terephthalate container, and the container was stored at 4°C for 1 day, 2 days, 4 days, or 23 days under the illumination of fluorescent light.

[0041] When the illuminance of the light irradiated on the container was measured with a 51001 digital illuminometer (manufactured by Yokogawa Electric Corporation), it was 1490 lux. Also, when the light after passing through the wall material of the brown translucent container made of polyethylene terephthalate was measured with the above illuminometer, it was 548 lux. Therefore, the brown translucent container made of polyethylene terephthalate is a container with a light shielding rate of about 63%.

[0042] (2) Measurement of enzyme activity value in the enzyme-containing aqueous solution after storage The enzyme activity value (after storage) in each enzyme-containing aqueous solution stored in the above (1) was calculated by the same method as in Example 3.

[0043] (3) Calculation of residual activity in the enzyme-containing aqueous solution after storage From the enzyme activity value (before storage) in the enzyme-containing aqueous solution measured in Example 3 and the enzyme activity value (after storage) in the enzyme-containing aqueous solution calculated in the above (2), the residual activity (%) after storage in each enzyme-containing aqueous solution was calculated by the following formula (I). The results are shown in Tables 1 and 2. Note that Table 1 shows the results when a transparent container was used, and Table 2 shows the results when a translucent container was used.

[0044]

[0045] [Comparative Example 4] Evaluation of residual activity of fructosylpeptide oxidase in each aqueous solution after storage Except for using the enzyme-containing aqueous solution a of Comparative Example 1 or the enzyme-containing aqueous solution b of Comparative Example 2 instead of using the enzyme-containing aqueous solution A of Example 1 or the enzyme-containing aqueous solution B of Example 2, the enzyme activity values (after storage) in the enzyme-containing aqueous solution a of Comparative Example 1 and the enzyme-containing aqueous solution b of Comparative Example 2 were calculated by the same method as in (1) and (2) of Example 4. Next, from the enzyme activity value (before storage) in each enzyme-containing aqueous solution measured in Comparative Example 3 and the enzyme activity value (after storage) in each enzyme-containing aqueous solution calculated as described above, the residual activity (%) after storage in each enzyme-containing aqueous solution was calculated by the above formula (I). The results are shown in Tables 1 and 2. Note that Table shows the results when a transparent container was used, and Table 2 shows the results when a translucent container was used.

[0046]

[0047]

[0048] The residual activity (%) after storage in each enzyme-containing aqueous solution is a value that indicates the percentage change in the enzyme activity value (after storage) in each enzyme-containing aqueous solution, based on the enzyme activity value (before storage) in each enzyme-containing aqueous solution. Therefore, enzyme-containing aqueous solutions in which enzyme activity is stably maintained will have a residual activity (%) close to 100% even after storage, while unstable enzyme-containing aqueous solutions will have a low residual activity (%). Furthermore, if the residual activity (%) in the enzyme-containing aqueous solution containing an additional compound is higher than that in the enzyme-containing aqueous solution a without the additional compound (sodium azide, tryptophan, or histidine), it can be concluded that the fructosyl peptide oxidase is stabilized by the additional compound.

[0049] As shown in Table 1, when using transparent containers, the residual activity after 23 days of storage was 71% and 49% for enzyme-containing aqueous solution A containing sodium azide and enzyme-containing aqueous solution B containing tryptophan, respectively. In contrast, the residual activity after 23 days of storage was very low, at 14% and 18% for enzyme-containing aqueous solution a without additional compounds such as sodium azide and enzyme-containing aqueous solution b containing histidine. Therefore, it was revealed that in the presence of a chromogen that produces methylene blue, fructosyl peptide oxidase is stabilized by the presence of sodium azide or tryptophan.

[0050] Furthermore, as shown in the results in Table 2, when using a translucent container, the residual activity after 23 days of storage was 102% and 100% for enzyme-containing aqueous solution A containing sodium azide and 100% for enzyme-containing aqueous solution B containing tryptophan, respectively. In contrast, the residual activity after 23 days of storage was 92% and 92% for enzyme-containing aqueous solution a without additional compounds such as sodium azide and 100% for enzyme-containing aqueous solution b containing histidine. From the above, it became clear that even when using a translucent container with a light-shielding rate of approximately 63%, fructosyl peptide oxidase is stabilized in the presence of sodium azide or tryptophan in the presence of DA-67, a chromogen that produces methylene blue.

[0051] [Example 5] Preparation of aqueous solutions containing fructosyl peptide oxidase Enzyme-containing aqueous solutions C and D were prepared in the same manner as in Examples 1 and 2, except that "methylene blue 1.3 mg / L" was added to enzyme-containing aqueous solution A from Example 1 and enzyme-containing aqueous solution B from Example 2, respectively. Note that "methylene blue 1.3 mg / L" is the amount assumed to be when approximately 5% of "DA-67 26.0 mg / L" in each enzyme-containing aqueous solution is converted to methylene blue.

[0052] [Comparative Example 5] Preparation of aqueous solutions containing fructosyl peptide oxidase. Except for adding an additional 1.3 mg / L of methylene blue to aqueous solution a of Comparative Example 1 and aqueous solution b of Comparative Example 2, enzyme-containing aqueous solutions c and d were prepared in the same manner as in Comparative Examples 1 and 2. The 1.3 mg / L of methylene blue is the amount assumed to be when approximately 5% of the 26.0 mg / L of DA-67 in each enzyme-containing aqueous solution is converted to methylene blue.

[0053] [Example 6] Evaluation of residual activity of fructosyl peptide oxidase in each aqueous solution after storage. Except for using enzyme-containing aqueous solution C or enzyme-containing aqueous solution D from Example 5 instead of enzyme-containing aqueous solution A from Example 1 or enzyme-containing aqueous solution B from Example 2, and using only transparent polystyrene containers as the containers for filling each enzyme-containing aqueous solution, the residual activity (%) after storage of enzyme-containing aqueous solution C and enzyme-containing aqueous solution D from Example 5 was calculated using the same method as in Examples 3 and 4. The results are shown in Table 3.

[0054] [Comparative Example 6] Evaluation of the residual activity of each aqueous solution after storage The residual activity (%) of each of the enzyme-containing aqueous solution c and enzyme-containing aqueous solution d of Comparative Example 5 was calculated in the same manner as in Examples 3 and 4, except that instead of using enzyme-containing aqueous solution A of Example 1 or enzyme-containing aqueous solution B of Example 2, enzyme-containing aqueous solution c or enzyme-containing aqueous solution d of Comparative Example 5 were used, and only transparent polystyrene containers were used as containers for filling each enzyme-containing aqueous solution. The results are shown in Table 3.

[0055]

[0056] When an enzyme-containing aqueous solution is stored, the chromogen that produces methylene blue in the solution deteriorates, causing methylene blue to be produced from the chromogen, and the concentration of methylene blue in the enzyme-containing aqueous solution to increase. Therefore, if fructosyl peptide oxidase is stabilized in each enzyme-containing aqueous solution to which methylene blue has been added in addition to DA-67, which is a chromogen that produces methylene blue, then it can be evaluated that fructosyl peptide oxidase is stabilized even when methylene blue is present in the enzyme-containing aqueous solution due to the deterioration of the chromogen that produces methylene blue.

[0057] Table 3 shows that when methylene blue was added to each enzyme-containing aqueous solution in addition to DA-67, a chromogen that produces methylene blue, the residual activity after 23 days of storage was 51% and 47% for enzyme-containing aqueous solution C containing sodium azide and enzyme-containing aqueous solution D containing tryptophan, respectively. In contrast, the residual activity after 23 days of storage was very low, at 1% and 7% for enzyme-containing aqueous solution c without additional compounds such as sodium azide and enzyme-containing aqueous solution d containing histidine. Therefore, it became clear that even in the presence of methylene blue, i.e., when methylene blue is present in the enzyme-containing aqueous solution due to the deterioration of the chromogen that produces methylene blue, the presence of sodium azide or tryptophan stabilizes fructosyl peptide oxidase.

[0058] From the above, it was found that by preparing an aqueous solution containing fructosyl peptide oxidase, methylene blue or a chromogen that produces methylene blue, and at least one compound selected from the group consisting of sodium azide and tryptophan, the fructosyl peptide oxidase in the aqueous solution can be stabilized and the fructosyl peptide oxidase in the aqueous solution can be stably stored.

Claims

1. A method for stabilizing fructosyl peptide oxidase in the presence of methylene blue or a methylene blue-producing chromogen, comprising the step of preparing an aqueous solution containing fructosyl peptide oxidase, methylene blue or a methylene blue-producing chromogen, and at least one compound selected from the group consisting of sodium azide and tryptophan.

2. The method according to claim 1, further comprising the step of storing the aqueous solution.

3. A method for preserving fructosyl peptide oxidase in the presence of methylene blue or a methylene blue-producing chromogen, comprising the step of preserving an aqueous solution containing fructosyl peptide oxidase, methylene blue or a methylene blue-producing chromogen, and at least one compound selected from the group consisting of sodium azide and tryptophan.

4. The method according to any one of claims 1 to 3, wherein the chromogen that produces methylene blue is a phenothiazine-based chromogen.

5. The method according to claim 4, wherein the phenothiazine-based chromogen is 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt.

6. The method according to any one of claims 1 to 3, wherein the concentration of at least one compound selected from the group consisting of sodium azide and tryptophan in the aqueous solution is 0.01 g / L to 20 g / L.

7. The method according to any one of claims 1 to 3, wherein the concentration of methylene blue in the aqueous solution is 0.01 mg / L to 10 mg / L, or the concentration of the colorant that produces methylene blue in the aqueous solution is 1 mg / L to 50 mg / L.

8. The method according to any one of claims 1 to 3, wherein the aqueous solution is contained in a container with a light-shielding rate of 70% or less.

9. The method according to claim 2 or 3, wherein the storage period in the step of storing the aqueous solution is 1 day to 2 years.

10. The method according to any one of claims 1 to 3, wherein the aqueous solution does not contain a proteolytic enzyme.

11. An aqueous solution containing fructosyl peptide oxidase, methylene blue or a chromogen that produces methylene blue, and at least one compound selected from the group consisting of sodium azide and tryptophan.

12. The aqueous solution according to claim 11, wherein the chromogen that produces methylene blue is a phenothiazine-based chromogen.

13. The aqueous solution according to claim 12, wherein the phenothiazine-based chromogen is 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt.

14. The aqueous solution according to any one of claims 11 to 13, wherein the concentration of at least one compound selected from the group consisting of sodium azide and tryptophan is 0.01 g / L to 20 g / L.

15. The aqueous solution according to any one of claims 11 to 13, wherein the concentration of methylene blue is 0.01 mg / L to 10 mg / L, or the concentration of the colorant that produces methylene blue is 1 mg / L to 50 mg / L.

16. An aqueous solution according to any one of claims 11 to 13, contained in a container with a light-shielding rate of 70% or less.

17. An aqueous solution according to any one of claims 11 to 13, which does not contain a proteolytic enzyme.

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