JP3362509B2 - Mannitol measurement method - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a measuring method utilizing an enzymatic reaction, and particularly to a measuring method for mannitol.

[0002]

2. Description of the Related Art Compared to a detection method using a chemical reaction, an assay method using an enzyme reagent does not use a dangerous reagent such as an acid, a base or an organic solvent, and a complicated reaction operation such as heating or extraction. It is a safe and environmentally friendly analysis method because it does not need to be performed and there is almost no need to consider the treatment of waste liquid.
In addition, the enzyme has the advantage that it has a high substrate specificity, excellent selectivity, and mild reaction conditions.

Therefore, various analytical methods utilizing enzyme reactions have been known. For example, a method of detecting reduced oxygen or increased hydrogen peroxide by the electrode method using the reaction of the substrate oxidant and hydrogen peroxide generated by the action of the oxidase from the substrate and oxygen, or the generated hydrogen peroxide Generally, the colorimetric method is used for quantification. In addition, in the measurement method using dehydrogenase, the coenzyme is added to the substrate and the redox reaction between the substrate and the coenzyme occurs due to the action of dehydrogenase, and the amount of coenzyme is increased or decreased by the colorimetric method. The method of detection is common.

When the final detection means is a colorimetric method among these methods, there are problems such as turbidity and coloring of the sample hindering accurate measurement, and complicated pretreatment for removing these. It was

Further, the measuring method of detecting with an electrode using an oxidase is not affected by turbidity or a coloring substance, but the kinds of oxidases having a sufficient activity suitable for practical use are limited, and thus various methods are used. We were unable to meet the measurement request. Regarding dehydrogenase, for example, alcohol dehydrogenase does not act much on methanol as compared with ethanol, and therefore is used when measuring ethanol in a system in which methanol is mixed. However, it is difficult to reproducibly measure nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD) used as a coenzyme of dehydrogenase by the electrode method, and dehydrogenase cannot be simply used. .

Among various objects to be measured, some are difficult to measure by detection by chemical reaction. For example, mannitol is a fructose-reduced sugar alcohol, and is contained in algae such as onions, mushrooms, and kelp. Examples of the use of mannitol include low-calorie sweeteners, food and cosmetic quality improvers, stabilizers, and the like. Therefore, the demand for measuring mannitol is increasing, but it is difficult to selectively measure only mannitol by a chemical reaction utilizing the properties of sugar alcohol in a system in which other sugar alcohols and sugars coexist. In the case of containing such various sugars and sugar alcohols, it is necessary to perform pretreatment and then measure by HPLC (high performance liquid chromatography) or the like, which is not suitable for simply measuring mannitol.

As described above, in order to selectively measure only mannitol, a measuring method using an enzyme is preferable, and a mannitol dehydrogenase can be used, but even a reaction using a mannitol dehydrogenase is possible. If the final detection means is a colorimetric method, there is the above problem.

[0008]

DISCLOSURE OF THE INVENTION The present invention provides a small number of types of enzymes having sufficient activity in conventional electrochemical measurements using only oxidases, and the measurement target is limited.
Provided is a method for solving the above problems and capable of measuring mannitol accurately and easily.

[0009]

The present invention includes, but is not limited to, the following aspects.

(1) A sample which may contain mannitol,
Mannitol dehydrogenase that catalyzes the reaction of producing fructose and a coenzyme reductant from the oxidant of mannitol and a coenzyme (nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate), the coenzyme oxidant, and the coenzyme reduction In a reaction system containing a substrate deoxidant Yred that produces a substrate reductant Yred and a coenzyme oxidant from the body and a substrate oxidant Yox of the second dehydrogenase, and a substrate oxidant Yox of the second dehydrogenase, Step A for producing a substrate-reduced Yred of the second dehydrogenase, and the produced Yred
Method B for measuring mannitol in a sample.

(2) The step of measuring the produced substrate reductant Yred is brought into contact with an immobilized oxidase that catalyzes the oxidation reaction of Yred, and the amount of change in the electrode active substance that decreases or increases is electrochemically measured. The measuring method according to (1), which comprises detecting.

(3) Furthermore, Y originally present in the sample
A sample obtained in the step C from the measured value of the substrate reductant Yred obtained by performing the step B after the step A for producing the substrate reductant Yred of the second dehydrogenase, which has a step C for measuring red The measuring method according to (1), in which the amount of Yred originally present in the sample is corrected. (4) The measuring method according to (2), wherein the substance that decreases or increases due to the reaction of the oxidase is oxygen or hydrogen peroxide.

(5) In mannitol assay according to (1), the second dehydrogenase is L-lactate dehydrogenase, its substrate oxidant Yox is pyruvate, and its substrate oxidant Yred is L-lactic acid. Method. (6) The method for measuring mannitol according to (2), wherein Yred is L-lactic acid and the oxidase is L-lactate oxidase.

(7) A sample which may contain mannitol,
Mannitol dehydrogenase, coenzyme oxidant C _ox , coenzyme reductant Cred and substrate oxidant Yox of the second dehydrogenase
In the reaction system containing the substrate reductant Yred and the second dehydrogenase that produces the coenzyme oxidant C _ox , and the substrate oxidant Yox of the second dehydrogenase, the substrate reductant of the second dehydrogenase Y
Step A for producing red and the produced substrate reductant Y
Method B for measuring red, the method for measuring mannitol in a sample.

[0015]

In the present invention, as the step A, the reaction of the mannitol dehydrogenase and the reaction of another second dehydrogenase are combined,
An amount of Yred corresponding to the amount of mannitol in the sample is produced. First, the reaction formula of mannitol dehydrogenase is shown as follows. However, the coenzyme is nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD) or nicotinamide adenine dinucleotide phosphate (hereinafter NADP).
Abbreviated) and the coenzyme oxidant C _OX is NAD or NADP, and the reductant C red is NADH or NADP.
Illustrated by H.

Mannitol dehydrogenase reaction Mannitol + NAD (P) → fructose + NAD
(P) H

Since this reaction is reversible and the reaction apparently stops when the reaction equilibrium is reached, not all mannitol is converted to fructose. Therefore, the produced fructose or NAD
It is necessary to convert (P) H into another substance and shift the equilibrium to allow the reaction to proceed. Also this fructose or NA
Since the consumption reaction of D (P) H is not effective unless it is carried out at the same time as the reaction of mannitol dehydrogenase, it is considered to use an enzyme which can be reacted under the same conditions. And in this invention, the 2nd dehydrogenase which shares the coenzyme utilized for the reaction of a mannitol dehydrogenase is utilized. The analysis cost can be reduced by combining the reaction of reoxidizing the reduced coenzyme, recycling the coenzyme, and reducing the amount of expensive coenzyme used.

In the case of a dehydrogenase, the equilibrium state of the reaction is almost always the substrate reductant Yred and the coenzyme oxidant N.
It can be used because it is biased in the direction in which AD (P) is generated. A second dehydrogenase and its substrate oxidant Yox are further added to the above reaction system of mannitol dehydrogenase,
The reaction of the second dehydrogenase is combined.

Second dehydrogenase reaction Yox + NAD (P) H → Yred + NAD (P) In the present invention, mannitol and more than this mole of Yox are added, and 1 mole of Yred is added to 1 mole of mannitol.
Can be generated.

Next, in step B, the amount of mannitol in the sample can be determined by measuring the produced Yred. For quantifying Yred, a method using an oxidase that specifically acts on Yred is preferable. The oxidase oxidizes Yred into the substrate oxidant Zox in the presence of dissolved oxygen to produce hydrogen peroxide. Representative reactions of oxidases are illustrated below.

Oxidase reaction Yred + O ₂ → Zox + H ₂ O ₂ This oxidase reaction is a one-way reaction that produces hydrogen peroxide from oxygen, and even if Zox coexists, it does not affect the reaction. Even if NAD (P) and NAD (P) H coexist, there is no effect. The mannitol dehydrogenase and the second dehydrogenase do not need to be inactivated because they inhibit the reaction of oxidase or are not inhibited by the reaction of oxidase.

Methods for detecting the oxidase reaction include electrochemical detection and the like, which will be described later. In the present invention, the coenzyme oxidant NAD (P) is reduced to NAD (P) H by the reaction of mannitol dehydrogenase, and the second
Oxidized by the reaction of the dehydrogenase of the enzyme returns to NAD (P). Therefore, it seems that the coenzyme is not involved in the reaction, but it is recycled as an electron carrier. Therefore, although NAD (P) is added to the reaction system in the present invention, mannitol can be completely converted to Yred even if the amount of NAD (P) is smaller than the amount of mannitol in the sample.

In carrying out the measurement, the conversion coefficient between mannitol and the substrate reduced product Yred does not necessarily have to be 100%, and can be calculated according to a predetermined coefficient. In other words, the mannitol concentration can be determined kinetically by stopping the reaction until the 100% reaction is completed. However, it is not necessary to control the reaction time accurately by completing the reaction, which is practically preferable.

In order to achieve such reaction conditions,
All the mannitol cannot be oxidized unless the amount of the substrate oxidant Yox added to the reaction system is larger than the sample mannitol amount. When the sample contains a substrate reductant Yred in addition to mannitol, the amount of the substrate reductant Yred in the sample is first measured in step C, and then the conversion reaction from mannitol to Yred in the sample is performed. It is necessary to determine the amount of mannitol by measuring the difference between the two and the increase amount of Yred from the difference between the two.

Although the reactions of mannitol dehydrogenase and the second dehydrogenase proceed at the same time, the reaction of oxidase is carried out in a separate step, so that Zox produced by the oxidation of Yred may be the same as Yox. Further, mannitol does not react with Zox produced by oxidase because NAD (P) is added to the reaction system. Therefore, mannitol is not recycled.

In the present invention, the object to be measured is mannitol, but various substances can be combined with the second dehydrogenase, examples of which are shown in Table 1.

[0027]

[Table 1]

Depending on the type of second dehydrogenase selected, Y
ox is automatically determined, and at the same time, Yred and oxidase to be measured are also determined. By this method, the measurement of Yred (for example, D-glucose, L-lactic acid, etc.) using an oxidase,
It is also possible to continuously measure mannitol. As the second dehydrogenase, one that reacts with the same coenzyme as mannitol dehydrogenase, that is, NAD or NADP must be selected.

The types and origins of the mannitol dehydrogenase and the second dehydrogenase used in the present invention are not particularly limited. Further, the active amounts of the mannitol dehydrogenase and the second dehydrogenase are not particularly limited. However, if the amount of activity is too small, the reaction takes too long, and the amount of change per unit time is small, which may make measurement difficult. If the amount of activity is large, the reaction will be fast, but the analysis cost will increase. Therefore, practically 0.1 to 20 U / ml of mannitol dehydrogenase, and the second dehydrogenase varies depending on the type of dehydrogenase used, but it is 1 / 100th of the mannitol dehydrogenase with the indicated activity.
It is preferably about 100 times.

Similarly, with respect to NAD or NADP used for the measurement, the reaction proceeds even if a small amount is present, but the rate is slow. Then, if a large amount is present, the reaction becomes faster, but the analysis cost increases, so 0.1-20 mM
A degree is preferable. And the amount of the substrate oxidant Yox of the second dehydrogenase should be 1.0 to 5 times the upper limit of mannitol measurement.

Next, the step B for quantifying Yred produced by the reaction of the second dehydrogenase by utilizing the oxidase reaction will be specifically described. Of course, Yre originally existed in the sample
The process C for measuring d is the same as the method. The quantification of hydrogen peroxide produced by the reaction of the oxidase is carried out, for example, by a method using peroxidase and 2,2′-azino-di (3-ethylbenzthiazoline) -6-sulfonic acid or 4-aminoantipyrine (Trinder. Method) or the like to measure the absorbance in the visible region. The visible light absorbance measurement is less susceptible to turbidity than the ultraviolet light absorbance measurement used to quantify coenzymes. However, the measurement using a spectrophotometer is difficult to measure accurately when the sample is turbid or contains a coloring substance, and therefore pretreatment is required.

Compared with this method, the electrochemical measuring method in which reduced oxygen or generated hydrogen peroxide is converted into an electric current value by an electrode and measured is easy to operate and less susceptible to turbidity and coloring substances. It is an excellent detection means. As the oxygen electrode for measuring the consumed oxygen, various known electrodes such as galvanic type and Clark type can be used.

As a hydrogen peroxide electrode for measuring hydrogen peroxide produced, carbon, platinum, nickel,
It is possible to use a known one using palladium or the like and silver or the like on the cathode side. Generally, platinum is often used as the anode because of its low overvoltage and high sensitivity. An electrode having a selective permeation film such as a polysiloxane film, an acrylic resin film, a protein film, an acetyl cellulose film, an albumin film on the surface of the electrode is desirable from the viewpoint of removing obstacles.

It is also possible to measure with an electrode in which an electron carrier such as dichloroindophenol, potassium ferricyanide, benzoquinone, a so-called mediator or the like is interposed. As the electrode system, a two-electrode hydrogen peroxide electrode or oxygen electrode composed of a working electrode and a counter electrode can be used, but from the viewpoint of stability and accuracy, it is composed of a working electrode, a reference electrode and a counter electrode.
Electrodes are more desirable.

Although the solution-form oxidase can be used, the oxidase-immobilized one has advantages that the enzyme can be repeatedly used and the reaction conditions of the enzyme can be easily defined. Further, it is preferable to use the oxidase-immobilized body in combination with an electrode for detecting an increasing or decreasing amount of change in oxygen or hydrogen peroxide, etc., because Yred can be detected in one step in a short time.

The method for immobilizing the enzyme is not particularly limited, and an adsorption method, a chemical bonding method, an entrapping method, etc. can be used. Above all, a chemical bonding method that can form a strong immobilized body is preferable.
Carriers used for immobilization include diatomaceous earth, silica gel, glass beads, alumina, ceramics, carbon, activated carbon, molecular sieves, silicone rubber, cellulose,
Agarose, amino acid polymers, etc. can be used. As the chemical bonding method, a method in which an amino group is introduced on the surface of a carrier with an aminosilanization reagent and further formylation is performed using a polyfunctional aldehyde such as glutaraldehyde, and then an enzyme is brought into contact to immobilize it is preferable. Take as an example. As a form of the immobilized enzyme, a method of immobilizing it on a carrier and packing it in a reactor such as a column can be considered.

Further, a membrane in which an oxidase is fixed by a cross-linking agent such as glutaraldehyde, formaldehyde, succinyl aldehyde can be attached to the electrode and used. At the time of immobilizing in a film form, other proteins such as albumin, globulin and gelatin can be added to crosslink the oxidase. The buffer used for the measurement should be p suitable for the oxidase.
Any material that has a buffering capacity with H and does not have an electrochemical effect on the electrode may be used.

[0038]

The contents of the present invention will be described in more detail with reference to the following examples, but of course the present invention is not limited thereto.

Example 1 Mannitol dehydrogenase and NA were added to an aqueous solution of mannitol.
After D, L-lactate dehydrogenase and pyruvate (Y _OX ) were contacted and reacted for a certain period of time (step A), the produced L-lactic acid (Yred) was measured (step B). L-lactic acid measurement
The hydrogen peroxide produced was measured by a platinum electrode using an L-lactic acid measuring device described below.

(1) Method for producing L-lactate oxidase-immobilized column 150 mg of fire-resistant brick (30 to 60 mesh), which is calcined diatomaceous earth, is thoroughly dried, and 10% γ-aminopropyltriethoxysilane in anhydrous toluene solution is used. After being soaked in water for 1 hour, it is thoroughly washed with toluene and dried. After the aminosilane-treated carrier was immersed in 5% glutaraldehyde for 1 hour, it was washed thoroughly with distilled water and finally pH adjusted.
Replace with 7.0, 100 mM sodium phosphate buffer and remove this buffer as much as possible. This formylated refractory brick was brought into contact with 200 μl of a solution prepared by dissolving L-lactate oxidase (manufactured by Sigma) at a concentration of 50 units / ml in 100 mM sodium phosphate buffer having a pH of 7.0, and at 0 to 4 ° C. for 1 day. Leave and fix. A column having an inner diameter of 3.5 mm and a length of 30 mm is packed with this enzyme-immobilized carrier to give an L-lactic acid oxidase-immobilized column.

(2) Production of hydrogen peroxide electrode The side surface of a platinum wire having a diameter of 2 mm is coated with heat-shrinkable Teflon,
One end of the wire is smoothed with a file and a No. 1500 emery paper. Using this platinum wire as a working electrode, a 1 cm square platinum plate as a counter electrode, and a saturated calomel electrode as a reference electrode, 0.1M
Electrolyte in sulfuric acid at +2.0 V for 10 minutes. After that, the platinum wire was washed well with water and then dried at 40 ° C for 10 minutes.
It is immersed in an anhydrous toluene solution of 10% γ-aminopropyltriethoxysilane for 1 hour and then washed. 20 mg of bovine serum albumin (Fraction V, manufactured by Sigma) is dissolved in 1 ml of distilled water, and glutaraldehyde is added to the solution to make the concentration 0.2%. 5 μl of this mixed solution was quickly put on a platinum wire prepared in advance, and dried and cured at 40 ° C. for 15 minutes to form a selectively permeable membrane, which was used as a hydrogen peroxide electrode.

An Ag / AgCl reference electrode was used as the reference electrode, and a conductive pipe was used as the counter electrode.

(3) L-lactic acid measuring device L-lactic acid was measured by the flow type L-lactic acid measuring device shown in FIG. The buffer solution (1) is sent by the pump (2), and 5 μl of the sample is injected from the autosampler (3). From the L-lactic acid in the sample, hydrogen peroxide is produced by the L-lactate oxidase-immobilized column (5) in the thermostat (4), and the change in current value is detected and detected by the hydrogen peroxide electrode (6). It is detected by the device (7). Further signals can be sent to the computer (10).

The composition of the buffer solution is 100 mM sodium phosphate, 50 mM potassium chloride, 1 mM sodium azide, and has a pH of 7.0.

(4) Measuring method The final concentration in the reaction system is NAD 5 mM, pyruvic acid 10 m
M, mannitol dehydrogenase (manufactured by Sigma) 1 unit / ml, L-lactate dehydrogenase (manufactured by Boehringer) 10
Unit / ml, containing sodium phosphate, pH 7.5
, Mannitol 1 mM, mannitol 2 mM,
Each of the solutions containing mannitol 5 mM was reacted at 25 ° C. for about 60 minutes (step A).

A current value (nA) obtained by measuring 1 mM, 2 mM and 5 mM of L-lactic acid using the above-mentioned reaction solution and distilled water as a blank and using the L-lactic acid measuring apparatus described in (3) above. ) Was asked.

(5) Results The current values of the mannitol reaction solution and the L-lactic acid standard solution are shown in Table 2.

[0048]

[Table 2]

The respective calibration curves of L-lactic acid and mannitol are as follows. Here, r is a correlation coefficient, and Y is a current value (nA) when the concentration is X (mM). L-lactic acid calibration curve r = 0.9999 Y = 23.82X + 0.78 mannitol calibration curve r = 1.0000 Y = 23.74X-0.04

The calibration curves for L-lactic acid and mannitol are almost the same, and when the reaction proceeds, the oxidation of mannitol is completed and pyruvic acid is reduced to produce L-lactic acid. Therefore, when the amount of L-lactic acid after the reaction is measured,
Mannitol can be measured.

Example 2 Using the same L-lactic acid measuring apparatus as in Example 1, a mixed solution of L-lactic acid and mannitol was measured. (1) Measurement method A sample solution in which an L-lactic acid standard solution and a mannitol standard solution were mixed, and a reaction reagent containing an enzyme, a coenzyme and the like were mixed at a volume ratio of 1: 1 and reacted at room temperature of 25 ° C for 1 hour. . The composition of the reaction reagent is NAD 10 mM, pyruvate 20 mM, mannitol dehydrogenase (manufactured by Sigma) 2 units / ml, L
-Lactate dehydrogenase (Boehringer) 20 units /
The pH is 7.5 including ml and sodium phosphate.

[A] The sample as it is (Step C), and [B] the reaction liquid in which mannitol has been completely oxidized and pyruvic acid has been reduced (Step A and Step B) -L-lactic acid concentration was measured using a lactic acid measuring device. Since the sample solution [B] after the reaction was diluted 2-fold, the obtained measurement value was doubled, and the L-lactic acid concentration of the first sample solution [A] was subtracted to obtain the mannitol concentration.

(2) Results The concentrations of L-lactic acid and mannitol of each sample are shown in Table 3, and both L-lactic acid and mannitol could be measured accurately.

[0054]

[Table 3]

[0055]

By using the enzyme reaction of the present invention,
It has become possible to easily and accurately quantify mannitol. Further, the substrate reductant Yred of the second dehydrogenase can also be continuously measured by a series of operations. For example,
It has become possible to measure two components, mannitol and L-lactic acid, continuously and simply.

[Brief description of drawings]

FIG. 1 shows a flow-type L-lactic acid measuring apparatus used in Examples.

FIG. 2 shows the enzymatic reaction of the present invention.

[Explanation of symbols]

1 buffer tank 2 pumps 3 samplers 4 constant temperature bath 5 L-lactate oxidase-immobilized column 6 Hydrogen peroxide electrode 7 detector 8 single board computer 9 RS232C code 10 personal computer 11 Sampler control signal 12 Liquid feed pump control signal 13 waste liquid

Continuation of the front page (56) Reference JP-A-56-87853 (JP, A) JP-A-6-229981 (JP, A) JP-A-7-31461 (JP, A) JP-A-7-98295 (JP , A) JP-A-7-322897 (JP, A) JP-A-57-120853 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 27/416

Claims (3)

(57) [Claims] 1. A sample which may contain mannitol, mannitol dehydrogenase, coenzyme oxidant C _ox , coenzyme reductant Cred and substrate oxidant Yox of the second dehydrogenase from substrate reductant Yred and coenzyme oxidant. in the second dehydrogenase, and a second reaction system containing a substrate oxidant Yox dehydrogenase to generate the C _ox, a step of producing a substrate reductant Yred the second dehydrogenase, as well as generated A method of measuring mannitol in a sample, which comprises the step B of measuring the substrate reductant Yred. 2. The step of measuring the produced substrate reductant Yred is brought into contact with an immobilized oxidase that catalyzes the oxidation reaction of Yred, and the amount of change in the electrode active substance that decreases or increases is detected electrochemically. The measuring method according to claim 1, wherein 3. The method further comprises a step C for measuring Yred originally present in the sample, wherein the step B is performed after the step A for producing the substrate reductant Yred of the second dehydrogenase. 2. The measuring method according to claim 1, wherein the amount of Yred originally present in the sample obtained in the step C is corrected from the measured value of the substrate reduced product Yred.