JPH1084994A - Enzymatic determination method of trehalose and reagent for trehalose analysis - Google Patents

Abstract

(57) Abstract: After pretreating glucose contained in a trehalose-containing specimen using mutarotase, glucose oxidase and catalase so as not to affect the enzymatic quantitative reaction of trehalose, A method for enzymatically measuring trehalose, which comprises inhibiting trehalose activity by sodium azide, decomposing trehalose in a sample by trehalose phosphorylase, and quantifying generated glucose using glucose oxidase. [Effect] The measurement blank, which is present in the test solution, can be enlarged to eliminate the influence of glucose that hinders the enzymatic measurement of trehalose, and without performing blank measurement.
Trehalose can be measured simply and accurately.

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 an enzymatic measurement method of trehalose that can be accurately measured.

[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.

[0005]

An object of the present invention is to provide a method for enzymatically determining trehalose in a test solution,
An object of the present invention is to provide a method for enzymatically measuring trehalose, which enables simple and accurate simultaneous quantification of a plurality of specimens without separately quantifying glucose originally contained in the test solution.

[0006]

Means for Solving the Problems In view of the above problems, as a result of intensive studies, the present inventors have found that glucose present in a test solution is eliminated in advance, and then trehalose is decomposed by trehalose phosphorylase to produce glucose produced. It was found that if quantified by glucose oxidase, it was not necessary to carry out two or more measurements per sample, and that trehalose could be measured simply and accurately, thus completing the present invention.

That is, the present invention provides a method for treating catalase after pretreating glucose mixed in a trehalose-containing specimen with mutarotase, glucose oxidase and catalase so as not to affect the enzymatic quantitative reaction of trehalose. An enzymatic measurement method of trehalose, which comprises inhibiting the activity with sodium azide, decomposing trehalose in a sample with trehalose phosphorylase, and quantifying the generated glucose using glucose oxidase.

[0008] The present invention also relates to a pretreatment reagent for elimination of glucose containing at least mutarotase, glucose oxidase and catalase, and the detection of produced glucose containing at least trehalose phosphorylase, sodium azide, peroxidase, 4-aminoantipyrine and a hydrogen donor. It is a trehalose analysis reagent or a trehalose synthase-producing microorganism-researching reagent composed of a combination with a reagent.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
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 trehalose-producing ability, a microorganism cell,
Examples include, but are not limited to, animal tissue and plant tissue crushed liquid. 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.

In the present invention, first, glucose mixed in a trehalose-containing specimen is pretreated with mutarotase, glucose oxidase and catalase, and the glucose is eliminated so as not to affect the enzymatic quantitative reaction of trehalose. . FIG. 1A shows an example of a reaction process in this pretreatment.

When α-D-glucose is present in a sample, the α-D-glucose is converted to β-D-glucose by mutarotase.
It is converted to D-glucose. The converted β-D-glucose (if β-D-glucose is present in the sample from the beginning, further β-D-glucose) reacts with oxygen and water by glucose oxidase to give β-D-glucose.
Produces 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.

[0012] By performing such a pretreatment, glucose which is originally present in the test liquid is not involved in the method of measuring trehalose enzymatically using trehalose phosphorylase, and is therefore the target substance in the present invention. Trehalose can be measured directly with high sensitivity.

In this pretreatment, mutarotase, glucose oxidase and catalase may be used sequentially, or a reagent containing mutarotase, glucose oxidase and catalase (a pretreatment reagent for eliminating glucose) may be used. This glucose elimination pretreatment reagent contains at least mutarotase, glucose oxidase and catalase, and a phosphate buffer or the like can be used as a solvent. In addition to these, bovine serum albumin and a surfactant may be contained for stabilizing and activating the enzyme.

The contents of mutarotase, glucose oxidase and catalase in the pretreatment reagent for eliminating glucose are 2 to 40 units / ml and 100 to 500 units / ml, respectively.
And 2000 to 5000 units / ml.

[0015] In the present invention, the enzymatic quantification reaction of trehalose is then performed. Catalase used in the above pretreatment is:
Since hydrogen peroxide used for the determination of glucose is decomposed, it is necessary to inhibit the enzyme activity. Sodium azide can be used to inhibit catalase activity. The amount of sodium azide used is preferably 0.1 to 0.5% by weight, particularly preferably about 0.1% by weight, per 1000 units / ml of catalase.

In the enzymatic quantification reaction of trehalose, trehalose in a sample is decomposed by trehalose phosphorylase, and the produced glucose is quantified using glucose oxidase. The quantification method is not particularly limited, but colorimetric quantification by the so-called glucose oxidase method can be performed. An example of the reaction process is shown in FIG.
Shown in

Trehalose in the specimen is first reacted 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. The mutarotase and glucose oxidase in this reaction process can be used as they are in the pretreatment, but are 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.

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.

In the present treatment step, sodium azide, trehalose phosphorylase, mutarotase, glucose oxidase, peroxidase, 4-aminoantipyrine and a hydrogen donor may be used sequentially (for sodium azide, treatment with glucose oxidase may be used). In any order, sodium azide, trehalose phosphorylase, mutarotase, glucose oxidase, peroxidase,
A product glucose detection reagent containing 4-aminoantipyrine and a hydrogen donor or a product glucose detection reagent containing sodium azide, trehalose phosphorylase, peroxidase, 4-aminoantipyrine and a hydrogen donor may be used. Preferably, a product glucose detection reagent containing sodium azide, trehalose phosphorylase, peroxidase, 4-aminoantipyrine and a hydrogen donor is used.

These formed glucose detection reagents can contain a phosphate buffer or the like as a solvent, and the phosphate can be supplied to the reaction with trehalose by trehalose phosphorylase. In addition to the 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, Nikko Chemical's O.D.
P10 and BT7 manufactured by Nikko Chemical Co., Ltd. may be included.

The contents of sodium azide, trehalose phosphorylase, peroxidase, 4-aminoantipyrine and hydrogen donor in the formed glucose detection reagent are 0.1 to 0.5% by weight and 0.055 to 0.11 unit / m, respectively.
l, 10-100 units / ml, preferably 0.5-5 mM and 0.5-5 mM.

The combination of the above-mentioned glucose elimination pretreatment reagent and the produced glucose detection reagent is useful as a trehalose analysis reagent or a trehalose synthase-producing microorganism-researching reagent. The enzymatic measurement of trehalose using the glucose elimination pretreatment reagent and the produced glucose detection reagent can be performed, for example, as follows.

First, a test solution and a pretreatment reagent for elimination of glucose are mixed, and incubated at a constant temperature, for example, 37 ° C., for a certain time, for example, for 20 minutes or more, to eliminate glucose in the test solution. . The test solution thus pretreated and the produced glucose detection reagent are mixed, and incubated at a constant temperature, for example, 37 ° C. for a certain time, for example, 5 minutes or more, and the result of the color reaction is obtained. 542
It is measured by absorbance near 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).
The measurement according to the present invention is simple in operation and is very suitable for automatic analysis (automatic method).

According to the present invention described above, after the glucose in the test solution has been eliminated in advance, the enzymatic quantitative measurement of trehalose using trehalose phosphorylase can be carried out as it is. The operation is simple and the accuracy is high as compared with the case where two or more measurements are required for one sample to take. Therefore, the method and reagent for the enzymatic measurement of trehalose of the present invention are extremely suitable especially for screening for trehalose detection.

[0027]

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 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 present invention (method for use), 1 to 10 g / l (about 2.6 to 2 g / l) is used.
A quantification of trehalose at a concentration of 6 mM) can be performed.

Example 2 Determination 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 present invention (automatic method),
Quantification of trehalose at a concentration of 1 to 10 g / l (about 2.6 to 26 mM) can be performed.

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 present invention (method for use), even if glucose is present in the test solution, at least 5 g / l of glucose can be treated to eliminate the effect. Thus, quantification of trehalose can be performed accurately.

Example 4 Measurement of the Influence of Glucose: Auto Method 0 g / l, 0.5 g / l, 1 g / l, 1.5 g / l, 2 g / l, 2. g / l
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 present invention (automatic method),
Even when glucose is present in the test solution, if the glucose concentration is 1 g / l or less, the effect can be eliminated and the quantification of trehalose can be accurately performed.

[0033]

According to the present invention, according to the present invention,
The measurement blank can be enlarged, and the influence of glucose which hinders the enzymatic measurement of trehalose can be eliminated, and trehalose can be measured simply and accurately without performing blank measurement.

[Brief description of the drawings]

FIG. 1 is a diagram showing an example of a reaction process in the present invention. (a) shows the reaction process in the pretreatment,
(b) shows the enzymatic quantitative reaction process of trehalose.

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 (6)

[Claims] The present invention relates to a method for preparing a trehalose-containing sample, comprising the steps of: pretreating glucose contained in a trehalose-containing specimen with mutarotase, glucose oxidase and catalase so as not to affect an enzymatic quantitative reaction of trehalose; A method for enzymatically measuring trehalose, which comprises inhibiting trehalose in a sample with trehalose phosphorylase and quantifying generated glucose using glucose oxidase, while inhibiting the sodium by sodium. 2. In order to pretreat glucose mixed in a trehalose-containing specimen, at least mutarotase,
Using a glucose elimination pretreatment reagent containing glucose oxidase and catalase,
The method for measuring trehalose enzymatically according to claim 1. 3. A method for quantifying glucose produced by decomposing trehalose in a sample, comprising using a produced glucose detection reagent containing at least trehalose phosphorylase, sodium azide, peroxidase, 4-aminoantipyrine and a hydrogen donor, The method for measuring trehalose enzymatically according to claim 1, wherein glucose is colorimetrically determined by a color reaction of hydrogen peroxide. 4. The method according to claim 1, wherein the hydrogen donor is phenol, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-.
Anisidine, N-ethyl-N- (2-hydroxy-3-
Sulfopropyl) -m-toluidine, N-ethyl-N-
At least one selected from the group consisting of (2-hydroxy-3-sulfopropyl) aniline and derivatives thereof
4. The method for enzymatically measuring trehalose according to claim 3, wherein the method is a seed. 5. A combination of a glucose elimination pretreatment reagent containing at least mutarotase, glucose oxidase and catalase, and a produced glucose detection reagent containing at least trehalose phosphorylase, sodium azide, peroxidase, 4-aminoantipyrine and a hydrogen donor. A trehalose analysis reagent comprising: 6. A combination of a glucose elimination pretreatment reagent containing at least mutarotase, glucose oxidase and catalase, and a produced glucose detection reagent containing at least trehalose phosphorylase, sodium azide, peroxidase, 4-aminoantipyrine and a hydrogen donor. A reagent for searching a trehalose synthase-producing microorganism, comprising: