JPH1099098A - Reagent for analyzing trehalose - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a reagent to enlarge a measurement blank existing in a test solution to eliminate influence of glucose disturbing enzymatic measurement of trehalose and thereby enabling to simply and accurately measure trehalose without conducting blank measurement. SOLUTION: This reagent for analyzing trehalose comprises a glucose- eliminating pretreatment reagent containing at least 2-40units/ml of mutarotase, 100-500units/ml of glucose oxidase and 2000-5000units/ml of catalase and a produced glucose-detecting reagent containing at least 0.055-0.11unit/ml of trehalose phosphorylase, 0.1-0.5wt.% of sodium azide, 10-100units/ml of peroxidase, 0.5-5mM of 4-aminoantipyrine and 0.5-5mM of a hydrogen donor.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a reaction system for enzymatically determining trehalose in a test solution, which can be simply and easily performed without separately determining the amount of glucose originally contained in the test solution. The present invention relates to a trehalose analysis reagent capable of performing accurate measurement.

[0002]

2. Description of the Related Art Hitherto, several methods for quantifying trehalose have been known. One is to measure the total sugar amount by anthrone method or the like, measure the amount of reducing sugar such as glucose by a Nelson method or the like, and subtract the amount of the reducing sugar from the total sugar amount to obtain non-reducing sugar. This is a method for measuring trehalose. However, there are non-reducing sugars other than trehalose (for example, sucrose), and accurate quantification of trehalose cannot be performed by subtracting the reducing sugar amount from the total sugar amount. In addition, the measurement process is very complicated, not only requires time and labor, but also tends to cause errors.

As another method, there is a quantitative method by high performance liquid chromatography. However, it takes a long time to measure one sample and is not suitable for measuring a large number of samples in a short time. When measuring an unknown sample containing trehalose, if a substance having the same retention time as that of trehalose is contained, there is a disadvantage that a correct measurement value cannot be obtained. As another method, an enzymatic quantification method using trehalase is known. This method is based on the principle that trehalose is decomposed into two molecules of glucose by treating with trehalase, and the generated glucose is quantified. However, when glucose is included in the test subject in addition to trehalose from the beginning, it is necessary to measure only the amount of glucose separately and subtract that amount from the total glucose amount, which complicates the process. However, there is a disadvantage that there are many error factors.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a reagent for enzymatically determining trehalose in a test solution, in which the amount of glucose originally contained in the test solution is separately determined. It is an object of the present invention to provide a trehalose analysis reagent which is capable of simply and accurately simultaneously quantifying a plurality of samples.

[0005]

Means for Solving the Problems In view of the above problems, as a result of intensive studies, the present inventors have determined a reagent capable of eliminating glucose present in a test solution in advance and a reagent which decomposes trehalose and produces glucose. It has been found that, when used in combination with a reagent that can be used, the measurement of trehalose can be performed simply and accurately without the necessity of performing measurement twice or more for one sample, and completed the present invention.

That is, the present invention relates to mutarotase 2-40
Unit / ml, glucose oxidase 100-500 units / ml
And a glucose elimination pretreatment reagent containing at least 2,000 to 5,000 units / ml of catalase, 0.055 to 0.11 units / ml of trehalose phosphorylase, and 0.1% of sodium azide.
~ 0.5 wt%, peroxidase 10-100 units / ml, 4-
Aminoantipyrine 0.5-5 mM and hydrogen donor 0.5-
A trehalose analysis reagent comprising a combination with a produced glucose detection reagent containing at least 5 mM.

Further, the present invention is the above-mentioned reagent for trehalose analysis, wherein the pretreatment reagent for eliminating glucose further comprises a phosphate buffer and bovine serum albumin. Further, the present invention is the above trehalose analysis reagent, wherein the produced glucose detection reagent further contains a phosphate buffer, ethylenediaminetetraacetic acid, a surfactant and bovine serum albumin.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The trehalose analysis reagent of the present invention comprises a combination of a glucose elimination pretreatment reagent (I) and a produced glucose detection reagent (II). The glucose elimination pretreatment reagent (I) contains at least mutarotase, glucose oxidase and catalase, and a phosphate buffer or the like can be used as a solvent. In addition, it may contain bovine serum albumin for stabilizing the enzyme, and a surfactant for activating the enzyme. It is preferable to use a phosphate buffer as a solvent, and preferably to contain bovine serum albumin.

The glucose-elimination pretreatment reagent (I) has a mutarotase content of 2 to 40 units / ml, a glucose oxidase content of 100 to 500 units / ml, and a catalase content of 2000 to 5000 units. Unit / ml.
By adjusting the content of mutarotase to 2 to 40 units / ml and the content of glucose oxidase to 100 to 500 units / ml, glucose (α-D
-Glucose, β-D-glucose) can be reliably eliminated. In addition, the content of catalase is 2000-5000
By setting the unit / ml, it is possible to reliably decompose hydrogen peroxide which affects the enzymatic quantitative reaction of trehalose.

The produced glucose detection reagent (II) contains at least trehalose phosphorylase, sodium azide, peroxidase, 4-aminoantipyrine and a hydrogen donor. The produced glucose detection reagent (II) can contain a phosphate buffer or the like as a solvent, and the phosphoric acid can be supplied to the reaction with trehalose by trehalose phosphorylase. In addition to phosphate buffer, ethylenediaminetetraacetic acid for stabilizing trehalose phosphorylase; a surfactant for activating enzymes, particularly trehalose phosphorylase; bovine serum albumin for stabilizing enzymes; Surfactants, for example, OP10 manufactured by Nikko Chemical Co., Ltd., BT7 manufactured by Nikko Chemical Co., Ltd., etc. may be included. It is preferable to use a phosphate buffer as a solvent, and it is preferable to contain ethylenediaminetetraacetic acid, a surfactant and bovine serum albumin.

Examples of the hydrogen donor include phenol, N-ethyl-N- (2-hydroxy-3-sulfopropyl)-
m-anisidine (ADOS), N-ethyl-N- (2-
Hydroxy-3-sulfopropyl) -m-toluidine (TOOS), N-ethyl-N- (2-hydroxy-3
-Sulfopropyl) aniline (ALOS) and derivatives thereof such as N-ethyl-N- (2-hydroxy-3
-Sulfopropyl) -3-methoxyaniline and the like, and each can be used alone or in appropriate combination.

The content of trehalose phosphorylase in the produced glucose detection reagent (II) is 0.055-0.11 unit / ml, and the content of sodium azide is 0.1-0.5.
% By weight, the content of peroxidase is 10 to 100 units / ml, and the content of 4-aminoantipyrine is 0.5 to 100 units / ml.
5 mM and the hydrogen donor content is 0.5-5 mM.
By setting the content of trehalose phosphorylase to 0.055 to 0.11 unit / ml, trehalose in the sample can be reliably converted to α-D-glucose, and the content of sodium azide to 0.1 to 0.5% by weight. This can sufficiently inhibit the enzymatic activity of catalase used in the glucose elimination pretreatment reagent (I). Further, by setting the content of peroxidase to 10 to 100 units / ml, the content of 4-aminoantipyrine to 0.5 to 5 mM, and the content of hydrogen donor to 0.5 to 5 mM, β-
D-glucose, and thus trehalose, can be accurately quantified.

When the trehalose analysis reagent of the present invention is used, the reaction proceeds as shown in FIG. Examples of the trehalose-containing specimen (test liquid) that can be measured in the present invention include a reaction liquid for screening a microorganism having a trehalose-producing ability, a crushed liquid of microbial cells, animal tissues, and plant tissues. However, the present invention is not limited to these. Glucose present in the sample from the beginning can be an interfering factor in the enzymatic quantification reaction of trehalose, but the sample in the present invention may be one that is expected to contain glucose in addition to trehalose, It may not be done.

(A) Use of Glucose Elimination Pretreatment Reagent (I) When α-D-glucose is present in the sample, the α-D-glucose
D-glucose is converted to β-D-glucose by mutarotase. Converted β-D-glucose (if β-D-glucose is present in the sample from the beginning,
Further, the β-D-glucose) reacts with oxygen and water by glucose oxidase to generate β-D-glucono-δ-lactone and hydrogen peroxide. The hydrogen peroxide generated here also affects the enzymatic quantitative reaction of trehalose, and is converted to water and oxygen by catalase.
As described above, by performing the pretreatment using the glucose elimination pretreatment reagent (I), the glucose which is originally present in the test liquid is involved in the enzymatic measurement method of trehalose using trehalose phosphorylase. Therefore, trehalose, which is the target substance, can be directly measured with high sensitivity.

(B) Use of Product Glucose Detection Reagent (II) The sodium azide in the product glucose detection reagent (II) is
Inhibits the enzyme activity of catalase used in the above pretreatment. Catalase degrades hydrogen peroxide used for the determination of glucose, so that glucose can be accurately determined by inhibiting its enzymatic activity. Trehalose in the sample first reacts with phosphoric acid by trehalose phosphorylase to produce α-D-glucose and glucose-1-phosphate. Next, the produced α-D-glucose is converted to β-D-glucose by mutarotase. The released and generated β-D-glucose reacts with oxygen and water by glucose oxidase to generate β-D-glucono-δ-lactone and hydrogen peroxide. As the mutarotase and glucose oxidase in this reaction process, those contained in the glucose elimination pretreatment reagent (I) can be used as they are, but not limited thereto.

The produced hydrogen peroxide reacts with 4-aminoantipyrine and a hydrogen donor by peroxidase to form a color.
By measuring the absorbance at 500 to 600 nm, desirably around 542 nm, β-D-glucose and thus trehalose can be quantified. The maximum absorption wavelength is 54
2 nm, and the wavelength that can be set by the automatic biochemical analyzer is 546
nm. The enzymatic measurement of trehalose using the trehalose analysis reagent of the present invention can be performed, for example, as follows.

First, a test solution and a pretreatment reagent for elimination of glucose
(I) and at a constant temperature, for example, at 37 ° C., for a certain time,
Incubate for 20 minutes or more, for example, to eliminate glucose in the test solution. The test solution thus pretreated and the produced glucose detection reagent (II) are mixed and incubated at a constant temperature, for example, at 37 ° C. for a certain time, for example, for 5 minutes or more, and a color reaction is performed. Is measured by the absorbance at around 542 nm.

This measurement can be generally performed by a manual method or an automatic method. Is a method in which a measurer puts a predetermined amount of a reagent at a predetermined time using a pipette or the like and measures it after a certain period of time. The auto method is a method generally used for clinical tests. This is a method of measuring using an automatic chemical analyzer (auto analyzer).
According to the trehalose analysis reagent of the present invention, the operation is simple and extremely suitable for automatic analysis (automatic method).

According to the trehalose analysis reagent of the present invention described above, after the glucose in the test solution is eliminated in advance, the enzymatic quantitative measurement of trehalose using trehalose phosphorylase can be performed as it is. Conventionally, the operation is simpler than the case where two or more measurements were required for one sample to obtain a sample blank.
Moreover, the accuracy is high. Therefore, the trehalose analysis reagent of the present invention is extremely suitable for screening for trehalose detection. The reagent of the present invention can also be used as a reagent for searching for a trehalose synthase-producing microorganism.

[0020]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. (Example 1) Determination of trehalose: method for use A reagent having the following composition was prepared.

Glucose elimination pretreatment reagent (R1) Phosphate buffer (pH 6.9) 200 mM mutarotase 10 units / ml Glucose oxidase 100 units / ml Catalase 2000 units / ml Bovine serum albumin 0.02% by weight produced glucose detection reagent (R2 ) Phosphate buffer (pH 6.9) 200 mM sodium azide 0.2 wt% ethylenediaminetetraacetic acid 2 mM 4-aminoantipyrine 2 mM N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline 2 mM Tween20 0.1% by weight peroxidase 10 units / ml trehalose phosphorylase 0.055 unit / ml bovine serum albumin 0.02% by weight

As a test solution, a trehalose aqueous solution containing 3 g / l of glucose (trehalose concentration: 0 g / l, 1 g / l)
l, 2g / l, 3g / l, 4g / l, 5g / l, 6g / l, 7g / l, 8g / l, 9g / l,
10 g / l). 50 μl of R was added to 50 μl of this test solution.
1 and incubate at 37 ° C for 20 minutes.
l of R2 was added and incubated at 37 ° C for exactly 20 minutes. The absorbance at 542 nm of this color reaction was measured. The results are shown in the graph of FIG. As shown in FIG. 2, according to the trehalose analysis reagent of the present invention (method for use), quantification of trehalose at a concentration of 1 to 10 g / l (about 2.6 to 26 mM) can be performed.

(Example 2) Quantification of trehalose: Auto method A reagent similar to that of Example 1 was prepared except that the concentration of trehalose phosphorylase was 0.11 unit / ml. As a test solution, a trehalose aqueous solution containing 1 g / l of glucose (trehalose concentration: 0 g / l, 1 g / l, 2 g / l, 3 g / l, 4 g / l, 5 g / l)
1, 6 g / l, 7 g / l, 8 g / l, 9 g / l, 10 g / l). After adding 200 μl of R1 to 20 μl of the test solution and incubating at 37 ° C. for 5 minutes, 200 μl of R2 was added and incubating at 37 ° C. for 5 minutes. About this coloring reaction, 54
The absorbance at 6 nm was measured. The results are shown in the graph of FIG.
As shown in FIG. 3, according to the trehalose analysis reagent of the present invention (automatic method), 1 to 10 g / l (about 2.6 to 26 mM)
Of trehalose at a concentration of

Example 3 Measurement of the Influence of Glucose: Technique Used As test liquids, 0 g / l, 0.5 g / l, 1 g / l, 1.5 g / l, 2 g / l, 2.
The same measurement as in Example 1 was performed except that an aqueous trehalose solution containing 5 g / l, 3 g / l, 4 g / l, and 5 g / l of glucose (trehalose concentration: 5 g / l) was used. The results are shown in the graph of FIG.

As shown in FIG. 4, according to the trehalose analysis reagent of the present invention, even if glucose is present in the test solution, at least 5 g / l of glucose is treated. Trehalose can be accurately determined.

Example 4 Measurement of the Influence of Glucose: Auto Method As test liquids, 0 g / l, 0.5 g / l, 1 g / l, 1.5 g / l, 2 g / l, 2.
The same measurement as in Example 2 was performed except that an aqueous trehalose solution containing 5 g / l and 3 g / l of glucose (trehalose concentration: 5 g / l) was used. The results are shown in the graph of FIG.
As shown in FIG. 4, according to the trehalose analysis reagent (automatic method) of the present invention, even if glucose is present in the test solution, if the glucose concentration is 1 g / l or less, The influence can be eliminated and the quantification of trehalose can be performed accurately.

[0027]

According to the trehalose analysis reagent of the present invention, the measurement blank present in the test solution can be enlarged, and the influence of glucose which hinders the enzymatic measurement of trehalose can be eliminated, and blank measurement can be performed. Without this, trehalose can be measured simply and accurately.

[Brief description of the drawings]

FIG. 1 is a diagram showing a reaction process using a trehalose analysis reagent of the present invention. (a) is the glucose elimination pretreatment reagent
(I) shows the reaction process, and (b) shows the reaction process using the produced glucose detection reagent (II).

FIG. 2 is a graph showing quantification results (method) of trehalose in Example 1.

FIG. 3 is a graph showing the results of trehalose quantification (automatic method) in Example 2.

FIG. 4 is a graph showing the result (method) of measuring the effect of glucose in a test solution in Example 3.

FIG. 5 is a graph showing the result (automatic method) of measuring the effect of glucose in a test solution in Example 4.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Masaaki Yamagishi 5-17-12 Nishiogagawa, Yaizu City, Shizuoka Prefecture (72) Inventor Yoshiyuki Totsuka 2-14-2 Shimogawara, Shizuoka City, Shizuoka Prefecture (72) Inventor Kubota Goo 1004 Ikeda, Shizuoka City, Shizuoka Prefecture (72) Inventor Mao Oguchi 4-25-8 Toro, Shizuoka City, Shizuoka Prefecture (72) Inventor Tadashi Wada 25-1 St. Isshiki, Shizuoka City, Shizuoka Prefecture Dias Kawaguchi B201 (72) Person Takafumi Sano 1759-4 Minamimatsuno, Fujikawa-cho, Abara-gun, Shizuoka Prefecture

Claims (3)

[Claims] 1. Mutarotase 2 to 40 units / ml, glucose oxidase 100 to 500 units / ml and catalase 20
A glucose elimination pretreatment reagent containing at least 00-5000 units / ml, and trehalose phosphorylase 0.055-0.1.
11 units / ml, sodium azide 0.1-0.5% by weight, peroxidase 10-100 units / ml, 4-aminoantipyrine
A trehalose analysis reagent comprising a combination with a product glucose detection reagent containing at least 0.5 to 5 mM and a hydrogen donor of 0.5 to 5 mM. 2. The trehalose analysis reagent according to claim 1, wherein the glucose elimination pretreatment reagent further contains a phosphate buffer and bovine serum albumin. 3. The trehalose analysis reagent according to claim 1, wherein the produced glucose detection reagent further contains a phosphate buffer, ethylenediaminetetraacetic acid, a surfactant, and bovine serum albumin.