Enzyme reagent, reagent kit, and method for stabilizing enzyme

Abstract

To provide an enzyme reagent which can be more stably stored for a long period of time than conventional products, a reagent kit, and a method for stabilizing an enzyme.SOLUTION: The enzyme reagent includes a lyase enzyme and an anionic surfactant. Preferably, the anionic surfactant is sodium octanoate or sodium cholate. The lyase system enzyme may be a citrate lyase or a phenylglyoxylate decarboxylase. The concentration of the anionic surfactant is 0.3% (wt / v) or less.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an enzyme reagent, a reagent kit, and a method for stabilizing an enzyme. [Background technology]

[0002] One known analytical method for measuring the amount of a test substance contained in a biological sample, soft drinks, food, etc. involves causing an enzymatic reaction with the test substance and measuring the amount of a compound produced by the test substance or an added reagent being converted in the reaction system. For example, Patent Documents 1 and 2 disclose reagent kits using such a method. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2024-32168 [Patent Document 2] Japanese Patent Application Publication No. 2018-126127 Summary of the Invention [Problem to be solved by the invention]

[0004] Reagent kits are typically required to have a shelf life of at least six months, taking into account their distribution and storage period in testing facilities. Reagent kits that use a method involving enzymatic reactions with test substances contain enzymes, and if the enzymes in the kit are inactivated before use, accurate measurement results will be inaccurate. In the case of reagent kits intended for quantitative analysis, if the quantitative results are affected by enzyme inactivation, the amount of enzymes contained cannot be accurately determined, and therefore, measures based on the amount may also be inappropriate, for example, leading to inappropriate medical treatments based on the results. Therefore, preventing the decline in enzyme activity is important for long-term storage and stable quality.

[0005] The present invention has been made in view of the above circumstances, and aims to provide an enzyme reagent, a reagent kit, and a method for stabilizing an enzyme that can be stored more stably for a long period of time than conventional products. [Means for solving the problem]

[0006] In order to solve the above problems, the enzyme reagent, reagent kit, and enzyme stabilization method of the present invention employ the following means.

[0007] A first aspect of the present invention provides an enzyme reagent comprising a lyase enzyme and an anionic surfactant.

[0008] The lyase enzyme may be a carbon-carbon lyase. The lyase enzyme may be a citrate lyase or a phenylglyoxylate decarboxylase.

[0009] In the first aspect, the anionic surfactant is sodium octanoate or sodium cholate. The anionic surfactant may be sodium cholate.

[0010] In the first aspect, the concentration of the anionic surfactant is preferably 0.3% (wt / v) or less.

[0011] A second aspect of the present invention provides a reagent kit for quantifying citric acid in a sample, comprising the enzyme reagent according to the first aspect, wherein the lyase enzyme is citrate lyase.

[0012] In the second aspect, the reagent kit is a two-liquid mixed type including a first reagent containing NADH (nicotinamide adenine dinucleotide) and a second reagent containing the enzyme reagent, and at least one of the first reagent and the second reagent may contain malate dehydrogenase (MDH).

[0013] A third aspect of the present invention provides a reagent kit for quantifying phenylglyoxylic acid in a sample, comprising the enzyme reagent according to the first aspect, wherein the lyase enzyme is phenylglyoxylic acid decarboxylase.

[0014] In the third aspect, the reagent kit is a two-liquid mixed type including a first reagent containing NAD (nicotinamide adenine dinucleotide) and a second reagent containing the enzyme reagent, and at least one of the first reagent and the second reagent may contain benzaldehyde dehydrogenase.

[0015] A fourth aspect of the present invention provides a method for stabilizing an enzyme, comprising the step of adding an anionic surfactant to a lyase enzyme. [Effects of the Invention]

[0016] According to the present invention, by allowing a lyase enzyme and an anionic surfactant to coexist in the kit, the inactivation of the lyase enzyme can be suppressed compared to when an anionic surfactant is not included, thereby providing an enzyme reagent and a reagent kit that can be stored for a longer period than conventional ones. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows the formulation and test results of Test 1. [Figure 2] FIG. 1 shows the formulation and test results of Test 2. [Figure 3] FIG. 1 shows the formulation and test results of Test 3. [Figure 4] FIG. 1 shows the reagent composition for Test 4. [Figure 5] FIG. 1 shows the results of Test 4 (Day 0). [Figure 6] FIG. 1 shows the results of Test 4 (25° C. 1 w). DETAILED DESCRIPTION OF THE INVENTION

[0018] Hereinafter, an embodiment of the enzyme reagent, the reagent kit, and the method for stabilizing an enzyme according to the present invention will be described with reference to the drawings.

[0019] [Enzyme Reagents] The enzyme reagent according to this embodiment contains a lyase enzyme and an anionic surfactant.

[0020] The enzyme reagent may be in the form of a solution or powder. When the enzyme reagent is in powder form, it is dissolved in a neutral solution before use. "Neutral" means a pH of 6.0 to 8.0, preferably 6.2 to 8.0.

[0021] The lyase enzyme is preferably a carbon-carbon lyase. The lyase enzyme may be citrate lyase or phenylglyoxylate decarboxylase.

[0022] The concentration of the lyase enzyme in the enzyme reagent is not particularly limited, but is, for example, 0.1 U / mL to 10 U / mL. The concentration of the lyase enzyme in the enzyme reagent may be 0.3 U / mL to 5 U / mL. When the enzyme reagent is in powder form, the concentration of the lyase enzyme in the enzyme reagent can be set to 1 to 10 times, preferably 2 to 5 times, that of the liquid form.

[0023] The anionic surfactant may be an octanoate or a cholate. The anionic surfactant may be sodium octanoate or sodium cholate. Preferably, the anionic surfactant is sodium cholate.

[0024] The concentration (wt / v) of the anionic surfactant in the enzyme reagent is preferably 0.30% or less, more preferably 0.05% or more and 0.30% or less, and even more preferably 0.20% or more and 0.30% or less.

[0025] The enzyme reagent may contain a chelating agent, a buffer, a non-ionic surfactant, and the like.

[0026] Examples of the chelating agent that can be used include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), hydroxyethyliminodiacetic acid (HIDA), triethylenetetraminehexaacetic acid (TTHA), glycoletherdiaminetetraacetic acid (GEDTA), and diethylenetriaminepentaacetic acid (DTPA). EDTA is preferably used as the chelating agent included in the reagent kit for quantifying citric acid according to this embodiment.

[0027] The buffer may be any buffer as long as the optimal pH of the enzyme is within the buffer range. Examples of buffers include MES (2-morpholinoethanesulfonic acid), Bis-Tris (bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane), MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid), Bis-Trispropane (1,3-bis[tris(hydroxymethyl)methylamino]propane, 2,2'-(propane-1,3-diylbis(azanediyl))bis(2-(hydroxymethyl)propane-1,3-diol)), HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid), and Tris (trishydroxymethylaminomethane). The buffering agent may preferably be MES (2-morpholinoethanesulfonic acid), Bis-Tris (bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane), or MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid). The concentration of the buffer in the enzyme reagent may be 25 mM to 300 mM.

[0028] The nonionic surfactant may be Tween® 20, Tween® 80, Emulgen 123P, Emulgen 130K, Emulgen 147, Emulgen 150, Emulgen 1118S-70, Emulgen 220, Emulgen 320P, Emulgen 709, Pluronic® F-64, TERGITOL, polyethylene glycol monooleate (10E.O.), polyethylene glycol monomethyl ether 400, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (30) sorbitan tetraoleate, Briji 10, Briji 20, Nikkol GO-460V, and the like.

[0029] In the enzyme reagent of this embodiment, the addition of an anionic surfactant and the presence of the lyase enzyme can suppress the decrease in enzyme activity, thereby enabling the enzyme reagent to be stored for a longer period of time than an enzyme reagent that does not contain an anionic surfactant.

[0030] [Reagent kit] The enzyme reagent is suitable for use as a reagent kit for quantifying citric acid or phenylglyoxylic acid in samples, including, but not limited to, plasma, serum, urine, food, and soft drinks.

[0031] A reagent kit for quantifying citric acid in urine will be described below as a representative example of this embodiment.

[0032] The reagent kit is a two-liquid mixed type containing a first reagent and a second reagent. The first reagent and the second reagent are stored independently until immediately before use. The storage temperature is 0°C to 10°C, preferably 2°C to 8°C.

[0033] The reagent kit also contains malate dehydrogenase (MDH), which may be contained in at least one of the first and second reagents.

[0034] The first reagent contains NADH. Before use, the first reagent is dissolved in a solution exhibiting a pH of 7.0 to weakly alkaline. "Weakly alkaline" means a pH of 8.0 to 9.0. That is, "a solution exhibiting a pH of 7.0 to weakly alkaline" refers to a solution exhibiting a pH of 7.0 to 9.0. An example of a solution exhibiting a pH of 7.0 to weakly alkaline is Tris (trishydroxymethylaminomethane) at pH 8.6.

[0035] The first reagent may contain a metal salt. The metal salt is preferably a salt of a metal that generates a divalent cation. Specifically, the metal salt may be, but is not limited to, a magnesium salt, a zinc salt, an iron(II) salt, or the like.

[0036] The first reagent may contain a non-ionic surfactant, such as Tween (registered trademark) 20.

[0037] The first reagent may contain a buffer, such as Tris (trishydroxymethylaminomethane).

[0038] The first reagent may further contain lactate dehydrogenase (LDH). LDH reacts pyruvate with NADH to produce lactate and NAD. + It is a dehydrogenase that produces

[0039] The second reagent is the enzyme reagent described in the above embodiment. The enzyme reagent contains at least a lyase enzyme and an anionic surfactant. The second reagent may also contain a chelating agent, a buffer, a nonionic surfactant, etc.

[0040] The lyase enzyme is citrate lyase. When sold as a reagent kit, the concentration of citrate lyase can be about 0.1 U / mL to 10 U / mL, preferably 0.3 U / mL to 5 U / mL. When the second reagent (enzyme reagent) is in powder form, the concentration can be set to about 1 to 10 times, preferably 2 to 5 times, that of the liquid form.

[0041] The reagent kit can also be a measurement kit that includes calibrators and control substances used in absorbance measurements, disposable equipment used for mixing reagents, etc. By using such a kit, when measuring many samples, for example, to quantify citric acid in urine, the amount of work required for cleaning the equipment can be reduced, and the quantification can be performed more easily.

[0042] The quantification of citric acid using the above-mentioned reagent kit can be carried out in the following steps S1 to S7. Here, the description will be given assuming that malate dehydrogenase (MDH) is contained in the first reagent. (S1) A first reagent (first solution) containing NADH and malate dehydrogenase (MDH) is obtained. (S2) A second reagent (second solution) containing citrate lyase and sodium cholate is obtained. (S3) The first solution and the sample are mixed to obtain a sample mixture. (S4) The absorbance of the sample mixture is measured, and the concentration of NADH in the sample mixture is calculated. (S5) A second solution is added to the sample mixture to obtain an enzyme reaction solution. (S6) The absorbance of the enzyme reaction solution is measured, and the concentration of NADH in the reaction solution is calculated. (S7) The amount of citric acid in the sample is quantified based on the calculated NADH concentration from before the start of the citric acid reaction to the end of the reaction.

[0043] First, a first solution containing the sample (sample mixture) is mixed with a second solution containing citrate lyase, resulting in the production of oxaloacetic acid through an enzymatic reaction. Next, malate dehydrogenase (MDH) reacts the oxaloacetic acid with the NADH contained in the first solution. The amount of citric acid in the sample is quantified by measuring the change in the amount of NADH present in the reaction solution. If the reaction proceeds as expected, the number of moles of NADH consumed in the second reaction stage will be equal to the number of moles of citric acid present in the system. By calculating the molar amount of NADH consumed before and after the reaction, the number of moles of citric acid present in the system can be calculated, and the amount of citric acid in the sample can be quantified.

[0044] In the quantitative determination method using the reagent kit according to this embodiment, the degree of change in NADH in the system in the step of quantifying the amount of citric acid in the sample may be calculated by measuring the change in absorbance derived from NADH. As an example, the concentration of citric acid in the sample can be calculated and quantified by measuring the decrease in absorbance derived from NADH at around 340 nm from before the reaction with citrate lyase using a spectrophotometer.

[0045] The degree of change in NADH in the reaction solution can also be calculated by measuring the rate of change in the amount of NADH in the reaction solution, instead of measuring and calculating the amount of change as described above.

[0046] (Variation) The following describes the application of the enzyme reagent of the above embodiment to a reagent kit for quantifying phenylglyoxylic acid in a sample. The reagent kit for quantifying citric acid is a measurement system that utilizes the oxidation reaction of NADH, whereas the reagent kit for quantifying phenylglyoxylic acid is a measurement system that utilizes the reduction reaction of NAD. Like the reagent kit for quantifying citric acid, the reagent kit is a two-liquid mixed type containing a first reagent and a second reagent, and the first reagent and the second reagent are stored independently until immediately before use.

[0047] The first reagent contains at least NAD. Before use, the first reagent is dissolved in a solution exhibiting a pH of 6.0 to 8.0. Such a solution may be, for example, a potassium phosphate buffer solution of pH 6.0.

[0048] The first reagent may include a metal salt, a non-ionic surfactant, and a buffering agent.

[0049] The second reagent is the enzyme reagent described in the above embodiment. The enzyme reagent contains phenylglyoxylate decarboxylase and an anionic surfactant. The second reagent may also contain a buffer, a nonionic surfactant, etc.

[0050] At least one of the first reagent and the second reagent contains benzaldehyde dehydrogenase.

[0051] When sold as a reagent kit, the concentration of phenylglyoxylate decarboxylase can be about 0.1 U / mL to 10 U / mL, preferably 0.3 U / mL to 5 U / mL. When the second reagent (enzyme reagent) is in powder form, the concentration can be set to about 1 to 10 times, preferably 2 to 5 times, that of the liquid form.

[0052] The action and effect of the anionic surfactant in the enzyme reagent according to this embodiment will be described below.

[0053] [Test 1: Evaluation of stability of enzyme reagents] Samples of formulations A to Q shown in Figure 1 were prepared and heated at 25°C for one week. Citrate lyase activity (CL activity) was measured using the samples immediately after preparation and after heating, and the residual rate of CL activity was calculated.

[0054] The composition of the specimen is as follows: 1.0-5.0U / mL CL 200mM MES (pH 6.2) 0.05~4% (wt / v) surfactant

[0055] (CL activity measurement) 1.0 mL of the reaction solution in a cuvette was preheated at 25°C for 3 minutes. 0.020 mL of the enzyme-containing sample was added to the reaction solution, which was then mixed about four times. The absorbance change at 365 nm was recorded for 5 minutes using a spectrophotometer, and the absorbance change per minute was calculated from the linear portion (ΔAbs test For the blank, 0.020 mL of the enzyme diluent was added instead of the sample, and the procedure was carried out in the same manner as above (ΔAbs blank / min). The number of samples was measured in duplicate. The absorbance at 365 nm is derived from NADH.

[0056] The CL activity was calculated using the following formula.

number

[0057] The composition of the reaction solution is as follows: 50mM glycylglycine (pH 7.9) 5.0mM potassium citrate 2.0mM zinc chloride 0.5mM NADH 30U / mL MDH

[0058] The composition of the enzyme dilution solution is as follows: 50mM glycylglycine (pH 7.9) 0.05% BSA (bovine serum albumin)

[0059] (Type of surfactant) The surfactants used were as follows: Surfactant-1: Sodium cholate (anionic; formulations B-J, L, P, Q) Surfactant-2: Tween® 20 (non-ionic; formulations K and L) Surfactant-3: Decyltrimethylammonium bromide (cationic; Formulation M) Surfactant-4: Cetyltrimethylammonium bromide (cationic; Formulation N) Surfactant-5: Benzyldimethyltetradecylammonium chloride (cationic; Formulation O)

[0060] Pure water was used instead of a surfactant in Formulation A. As a control, the residual rate of CL activity in Formulation A to which pure water was added was 21%.

[0061] As shown in Figure 1, in formulations N and O, which contained a cationic surfactant, CL activity decreased significantly immediately after sample preparation. In formulation M, which also contained a cationic surfactant, CL activity was maintained at the level seen immediately after sample preparation, but after heating at 25°C for one week, the residual CL activity decreased to 13%.

[0062] In formulation K, which contained a nonionic surfactant, the CL activity was maintained immediately after sample preparation, but the residual CL activity decreased to 15% after heating at 25°C for one week.

[0063] On the other hand, in formulation C, in which an anionic surfactant was added, the residual CL activity after heating at 25°C for one week was 54%.

[0064] In formulation L, which contained a nonionic surfactant in addition to an anionic surfactant, the residual rate of CL activity was as high as 60%.

[0065] These results confirmed that cationic and nonionic surfactants had no enzyme stabilizing effect, while anionic surfactants contributed to enzyme stabilization. They also suggested that nonionic surfactants did not affect the stabilizing effect of anionic surfactants.

[0066] (enzyme concentration) The effect of changing the concentration of the enzyme (citrate lyase) on the residual rate of enzyme activity was examined (formulations D, P, and Q). The surfactant used in this test was sodium cholate (anionic).

[0067] As shown in Figure 1, when a certain amount of anionic surfactant was added to an enzyme reagent and heated, it was confirmed that the residual rate was the same for all formulations, regardless of the enzyme concentration.

[0068] This result suggests that the presence of a certain amount of anionic surfactant in the enzyme reagent is important for stabilizing the enzyme activity.

[0069] (anionic surfactant concentration) The effect of varying the concentration of anionic surfactant (0% to 4.00% (wt / v)) on the residual rate of enzyme activity was examined (formulations A to J). The surfactant used in this test was sodium cholate (anionic), and the enzyme was citrate lyase.

[0070] According to FIG. 1, the residual rates of the formulations (B to J) to which an anionic surfactant was added were all about 2 to 3.5 times higher than that of the formulation (A) to which an anionic surfactant was not added.

[0071] When the concentration (wt / v) of the anionic surfactant was between 0.05% and 0.30%, the residual CL activity increased in a concentration-dependent manner. In particular, when the concentration (wt / v) of the anionic surfactant was between 0.20% and 0.30%, the residual CL activity was approximately three times that of Formulation A.

[0072] On the other hand, when the sodium cholate concentration (wt / v) was 0.40% or higher, the reagent dissolved in the solution when it was prepared, but in an accelerated test assuming long-term storage, i.e., after heating, crystals and precipitates were confirmed to form in the solution.

[0073] These results confirmed that adding an anionic surfactant to an enzyme reagent at a concentration (wt / v) of 0.30% or less, preferably 0.05% to 0.30%, and more preferably 0.20% to 0.30%, is effective in stabilizing enzyme activity.

[0074] [Test 2: Types of anionic surfactants] Samples were prepared using different types of anionic surfactants and heated at 25°C for one week. As in Test 1, citrate lyase activity (CL activity) was measured using the samples immediately after preparation and after heating, and the residual CL activity was calculated.

[0075] The composition of the specimen is as follows: 2.0U / mL CL 50mM MES (pH 6.2) 0.10% (wt / v) anionic surfactant

[0076] The following anionic surfactants were used: Sodium octanoate Sodium cholate

[0077] As a control, the residual rate was also measured for a sample in which pure water was used instead of a surfactant.

[0078] The measurement results are shown in Figure 2. The residual rate of all samples containing anionic surfactants was higher than that of the control without added surfactant. In particular, the residual rate of CL activity in the sample with added sodium cholate was approximately two-fold higher than that of the control.

[0079] Similar tests were also conducted when sodium cholate, which had a high residual rate, was applied to another enzyme (phenylglyoxylate decarboxylase: PGDC). The results are shown in Figure 2. It was confirmed that when sodium cholate was applied, the residual rate of PGDC activity was more than 1.5 times higher than the control.

[0080] PGDC is a type of lyase enzyme, specifically classified as a carbon-carbon lyase. These results suggest that anionic surfactants are effective in stabilizing lyase enzymes.

[0081] PGDC activity was measured as follows. 0.967 mL of the reaction solution in a cuvette was preheated at 25°C for 3 minutes. 0.033 mL of the sample was added, and the mixture was gently mixed. The absorbance change at 340 nm was recorded for 5 minutes using a spectrophotometer, and the absorbance change per minute was calculated from the linear portion (ΔAbs test For the blank, 0.033 mL of the enzyme diluent was added instead of the sample, and the procedure was carried out in the same manner as above (ΔAbs blank / min). The number of samples was measured in duplicate because this was a simple evaluation. The absorbance at 340 nm is derived from NADH.

[0082] PGDC activity was calculated using the following formula:

number

[0083] The composition of the reaction solution is as follows: 100mM potassium phosphate buffer (pH 6.0) 1.0mM magnesium chloride 0.3mM NADH 0.5mM cocarboxylase 8.3mM phenylglyoxylic acid 0.25U / mL alcohol dehydrogenase

[0084] The composition of the enzyme dilution solution is as follows: 10mM potassium phosphate buffer (pH 6.0)

[0085] The composition of the sample containing PGDC is as follows: 1.3U / mL PGDC 50mM Tris (pH 6.0) 0.1% (wt / v) anionic surfactant

[0086] [Test 3: Effect of buffering agent] Samples were prepared using different buffer types and concentrations and heated at 25°C for one week. As in Test 1, citrate lyase activity (CL activity) was measured using the samples immediately after preparation and after heating, and the residual CL activity was calculated. The residual enzyme activity was measured in the same manner as in Test 1.

[0087] The buffers used were MES, Bis-Tris, and MOPSO.

[0088] The composition of the specimen is as follows: 2.0U / mL CL 25mM~300mM buffer (pH 6.2) 0.10% (wt / v) sodium cholate

[0089] The measurement results are shown in Figure 3. In Figure 3, the measurement results of the sample prepared using pure water (without sodium cholate) in Test 2 are also shown as a control for comparison.

[0090] Within the buffer concentration range of 25 mM to 300 mM, the higher the concentration, the higher the residual CL activity. When the buffer was MES, the residual CL activity exceeded 50% at concentrations of 100 mM to 300 mM. In other words, the residual CL activity was more than twice that of the control.

[0091] The residual rate of CL activity was higher than that of the control for all of the buffers shown in Figure 3. These results confirmed that buffers within the buffer range where the optimal pH of the enzyme used is within the buffer range do not inhibit the stabilizing effect of the anionic surfactant (sodium cholate).

[0092] [Test 4: Enzyme stability of the reagent kit] The enzyme reagent described in the above embodiment was assumed to be applied to a reagent kit for quantifying citric acid, and the stability of the enzyme was confirmed.

[0093] The reagent kit for citric acid quantification includes a first reagent (R1) and a second reagent (R2). The compositions of the first and second reagents are shown in Figure 4. To facilitate comparison with Test 1, Figure 4 indicates which formulation in Test 1 the second reagent corresponds to (Formulations A, D, K, L, and P).

[0094] (Sample preparation) Day 0: The first and second reagents were used for measurement immediately after preparation.

[0095] 25℃1w: After preparing the first and second reagents, the first reagent was stored at 4°C and the second reagent at 25°C for one week before being used for measurement.

[0096] (measurement) The prepared or stored reagents were loaded onto a general-purpose automated analyzer (Hitachi High-Tech, Hitachi Automated Analyzer 3500), and the sample, first reagent, and second reagent were mixed at a volume ratio of 0.6:40:10. The absorbance was measured over time. The absorbance derived from NADH was measured at approximately 37°C using a two-point end-point method with a dominant wavelength of 340 nm and a secondary wavelength of 405 nm.

[0097] Figure 5 shows the measurement results for Day 0. Figure 6 shows the measurement results for 25°C 1w. In Figures 5 and 6, the horizontal axis represents the elapsed time (minutes), with 0 minutes being the time immediately after mixing the first reagent and citric acid (sample), and the vertical axis represents absorbance (Abs. x 10000). The "0 minutes," "5 minutes," and "10 minutes" on the horizontal axis in Figures 5 and 6 represent the time immediately after mixing the first reagent and sample, the time when the second reagent was added to start the reaction, and 5 minutes after the start of the reaction, respectively.

[0098] According to Figure 5, the absorbance of the sample immediately after preparation (Day 0) changed similarly for all of the formulations A, D, K, L, and P. In other words, it was confirmed that the reaction rate was the same regardless of the formulation, and the reaction was completed 5 minutes after the start of the reaction.

[0099] On the other hand, in the sample using the second reagent after heating at 25°C for one week (25°C 1w) shown in Figure 6, it was confirmed that the reaction was completed 5 minutes after the start of the reaction for all of the formulations A, D, K, L, and P, but differences in the reaction rate were observed for each formulation.

[0100] The reaction rate of the sample of formulation A, which did not contain a surfactant, slowed down after the second reagent was heated at 25°C for one week. The reaction rate of the sample of formulation K, which contained a nonionic surfactant instead of an anionic surfactant, was significantly slower than that of formulation A.

[0101] In contrast, the reaction rate of the formulations (D, L, P) containing an anionic surfactant was similar to that on Day 0. These results confirmed that the storage stability of the reagent kit was improved by mixing the enzyme and anionic surfactant in the second reagent.

[0102] Since the reaction rate decrease was suppressed in formulations D and P, which have different enzyme concentrations, it was confirmed that the anionic surfactant has a stabilizing effect at least for enzyme concentrations of 1 U / mL to 3 U / mL and does not affect the quantification of citric acid.

[0103] Formula L differs from Formula D in that it contains a nonionic surfactant (Tween® 20), but in Figure 6, the absorbance profiles of Formula L and Formula D overlapped. This confirmed that the presence of a nonionic surfactant did not affect the enzyme stabilizing effect of the anionic surfactant.

Claims

1. An enzyme reagent comprising a lyase enzyme and an anionic surfactant.

2. 2. The enzyme reagent according to claim 1, wherein the lyase enzyme is a carbon-carbon lyase.

3. 2. The enzyme reagent according to claim 1, wherein the lyase enzyme is citrate lyase or phenylglyoxylate decarboxylase.

4. 2. The enzyme reagent according to claim 1, wherein the anionic surfactant is sodium octanoate or sodium cholate.

5. 2. The enzyme reagent according to claim 1, wherein the anionic surfactant is sodium cholate.

6. 2. The enzyme reagent according to claim 1, wherein the concentration of the anionic surfactant is 0.3% (wt / v) or less.

7. A reagent kit for quantifying citric acid in a sample, comprising the enzyme reagent according to claim 1, wherein the lyase enzyme is citrate lyase.

8. a first reagent containing NADH; a second reagent containing the enzyme reagent; 8. The reagent kit according to claim 7, which is a two-liquid mixing type comprising: the first reagent and the second reagent, and wherein at least one of the first reagent and the second reagent contains malate dehydrogenase (MDH).

9. A reagent kit for quantifying phenylglyoxylic acid in a sample, comprising the enzyme reagent according to claim 1, wherein the lyase enzyme is phenylglyoxylic acid decarboxylase.

10. a first reagent containing NAD; a second reagent containing the enzyme reagent; It is a two-component mixed type equipped with 10. The reagent kit according to claim 9, wherein at least one of the first reagent and the second reagent contains benzaldehyde dehydrogenase.

11. A method for stabilizing an enzyme, comprising the step of adding an anionic surfactant to a lyase enzyme.

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