JPH05192191A - Determination of dissolved oxygen concentration - Google Patents

Abstract

(57) [Summary] [Purpose] To provide a new method for measuring dissolved oxygen concentration. [Structure] Dissolved oxygen in a sample is reacted with 2- (4-hydroxyphenyl) glycine or 4-hydroxymandelic acid in the presence of an oxidase (for example, laccase derived from Coriolus versicolor IFO9791) to produce 4-hydroxybenzaldehyde. The dissolved oxygen concentration is measured by quantifying. The method of the present invention can measure the dissolved oxygen concentration even when the liquid volume of the sample is 100 to 200 μl.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel method for measuring dissolved oxygen concentration using a certain phenolic compound and an oxidase.

[0002]

2. Description of the Related Art The presence of molecular oxygen dissolved in water is essential for the growth of fish and aerobic microorganisms. If this dissolved oxygen is reduced, the fish and fish will die and aerobically. Microorganisms stop their growth and physiological action. For this reason,
In culturing and culturing these organisms, management of dissolved oxygen concentration is very important. Currently known methods for measuring dissolved oxygen concentration are roughly divided into chemical methods and electrochemical methods. The chemical methods include the Winker method, the Miller method, and their modified methods. In particular, the Miller method is an excellent method because it has few interfering substances that affect the measured values. On the other hand, the electrochemical method includes a polarographic method and a galvanic cell method, which are widely used because they have excellent stability.

[0003]

As described above, many methods have been proposed and widely used for measuring the dissolved oxygen concentration. However, all of these methods require a considerably large amount of sample to measure the dissolved oxygen concentration, so it is difficult to collect many kinds of samples at one time, especially when collecting samples in the field. there were. Therefore, a method of measuring dissolved oxygen that can be measured with a smaller amount of sample is desired.

[0004]

Means for Solving the Problems As a result of repeated research to solve the above-mentioned problems, the present inventors produce dissolved oxygen in a sample by reacting it with a certain phenol compound in the presence of an oxidase. The present invention has been completed by finding that the dissolved oxygen concentration in a sample can be accurately measured by quantifying 4-hydroxybenzaldehyde even with a small amount of sample.

The method for measuring dissolved oxygen concentration provided by the present invention is as shown in the following reaction formula, in which an oxidase and a phenol compound are added to a sample solution and the sample solution is subjected to an oxidative decarboxylation reaction catalyzed by the oxidase. The amount of 4-hydroxybenzaldehyde produced is proportional to the amount of dissolved oxygen in the product, and the produced 4-hydroxybenzaldehyde is quantified by a known method.

[Chemical 2] Usually, an oxidase and a phenol compound are dissolved in a buffer solution so that each has a certain concentration to prepare an oxidase solution and a phenol compound solution, which are added to a certain amount of sample solution, mixed, and reacted for a certain time. , 4-hydroxybenzaldehyde produced is quantified.

As the oxidase used in the method of the present invention, any one can be used regardless of its type or origin as long as it catalyzes the above reaction.
Bilirubin oxidase, peroxidase, ascorbate oxidase, ceruloplasmin, etc. can be used. Among them, a particularly preferable oxidase is Coriolus versicolor.
lac), lacquer obtained from lacquer, etc., and one example thereof is Coliora verscicolor IFO9791.
(Coriolus versicolor IFO9
The laccase derived from 791) can be mentioned. This strain is the List of Cultures (LIST OF CULTURES) No. 8 published by Fermentation Research Institute
Edition, Vol. 1 (1988), which is a known strain, and can be easily obtained.

The amount of oxidase added is not particularly limited. It may be appropriately selected so that the reaction is completed within a predetermined time. For example, when the reaction is completed in 1 hour, about 50-
It may be added in a concentration range of 200 U / ml.
The enzyme unit is defined as 50 mM phosphate buffer (pH
6.0), the amount of enzyme that oxidizes 1 μmol of syringaldazine in 1 minute at 30 ° C. was set to 1 U (unit) (E).
nzyme Microb. Technol. , 19
81, Vol. 3, p55-58). The phenol compound that can be used in the method of the present invention is 2- (4-hydroxyphenyl) glycine or 4-hydroxymandelic acid, and any of them can be preferably used. The amount of these compounds added may be an excess amount as compared with the amount of dissolved oxygen in the sample, and is usually 10 to 100 mM, preferably 40 to
It may be added so that the concentration becomes 80 mM.

The reaction temperature may be any temperature as long as the oxidative decarboxylation reaction catalyzed by the oxidase proceeds, and may be, for example, 20 to 60 ° C. Considering this, the temperature is preferably 25 to 35 ° C. Also, the pH of the reaction solution may be any pH as long as the above reaction proceeds, and for example, it can be set to pH 5 to 7, but considering the activity of oxidase and the like, pH 5.5 to 6.5. Is preferred. Regarding the reaction time, the reaction may be carried out for a sufficient time so that the oxidative decarboxylation reaction catalyzed by an oxidase sufficiently progresses and 4-hydroxybenzaldehyde corresponding to the amount of dissolved oxygen is quantitatively produced. For example, as shown in Example 1 which will be described later, the change over time of the amount of 4-hydroxybenzaldehyde produced is determined by previously determining other conditions such as the liquid amount of the sample, the amount of enzyme, and the pH, and the aldehyde is quantitatively determined. The time to generate can be set.

The method for quantifying 4-hydroxybenzaldehyde in the present invention may be any method as long as it has a detection sensitivity of 1 μM or more, and high performance liquid chromatography (HPLC) method and chemical color development method are preferably used. For example, in the high performance liquid chromatography (HPLC) method,
Octadecyl silanized silica gel (OD
S), and a system using water-containing methanol or water-containing acetonitrile to which a buffer is optionally added as a mobile phase. The separated 4-hydroxybenzaldehyde has a wavelength of 260 to 300 nm, preferably 280 to 2 nm.
It can be detected by absorption at 85 nm and quantified using an integrator.

Examples of the chemical color development method include a method utilizing a color reaction between 2,4-dinitrophenylhydrazine and an aldehyde. That is, the generated 4-
Hydroxybenzaldehyde and 2,4-dinitrophenylhydrazine are condensed under acidic conditions to form a hydrazone,
Further, by subjecting this to a quinoid structure exhibiting a red color under alkaline conditions, the absorbance at around 500 nm is measured. This method has a molar extinction coefficient of about 32 at 500 nm for the quinoid structure produced.
As large as 000, 4-hydroxybenzaldehyde can be quantified with extremely high sensitivity.

A major feature of the method of the present invention is that the dissolved oxygen concentration can be accurately measured even when the amount of the sample is small. If the sample has a liquid volume of 100 to 200 μl, it can be sufficiently measured, so that there is an advantage that a large number of samples can be collected outdoors. Further, since the value obtained by the method of the present invention is in good agreement with the value obtained by the conventional method,
It is easy to replace the conventional method with this method.

Hereinafter, the present invention will be described in more detail with reference to examples.

【Example】

Example 1 2-in 50 mM sodium phosphate buffer (pH 6.0)
(4-Hydroxyphenyl) glycine was dissolved at 50 mM to prepare a substrate solution. This substrate solution 1.2
Laccase in ml (Coliorus versicolor IF
O 9791-derived, specific activity 4.8 × 10 ³ U / mg, protein concentration 2.3 mg / ml) 6 μl was added and stirred to prepare a reaction solution. About 1 ml of this was sucked into a glass syringe (1.0 ml volume) equipped with a needle for a rheodyne injector so that air would not enter the syringe. Leave this syringe at room temperature (28 ° C) every 10 minutes,
Injected into the high performance liquid chromatography from Rheodyne injector, under the following conditions, 4-hydroxybenzaldehyde eluting at a retention time of 5.7 minutes, using an integrator by an absolute calibration curve method using a standard sample,
Automatic quantification was performed.

Column: YMC-Pack A-302S
-5 120A ODS (4.6 × 150 mm) Mobile phase: 40% (V / V) methanol / 0.02M phosphate buffer (pH 6.8) Flow rate: 0.5 ml / min Detection: UV280 nm Analytical sample volume: 10 μl

The results are shown in Table 1.

[Table 1] As shown in Table 1, the production amount of 4-hydroxybenzaldehyde increased until 60 minutes, but after that, it showed an almost constant value (0.626 μmol / ml). This indicates that the oxidative decarboxylation reaction of laccase was stopped because all the dissolved oxygen in the reaction liquid in the syringe was consumed. Therefore, in order to measure the amount of dissolved oxygen in the sample under this condition, the reaction time should be 60 minutes or longer.

Example 2 0.5 ml of the reaction solution prepared in the same manner as in Example 1 was sucked into a glass syringe (1.0 ml volume) equipped with a needle for a rheodyne injector so that air would not enter the syringe, Furthermore, the sample for measuring the dissolved oxygen concentration 0.
1 ml was aspirated. This was left to stand at room temperature (28 ° C.) for 60 minutes, and the concentration of 4-hydroxybenzaldehyde was quantified under the same conditions as in Example 1 to find that it was 0.587 μmol / m 2.
It was l. From this result, the dissolved oxygen concentration in the sample was calculated by the following calculation formula, and was found to be 0.392 in terms of oxygen atoms.
It was μmol O / ml. (0.587 μmol / ml × 0.6 ml−0.626 μmol / ml × 0.5 ml) × 10 = 0.392 μmol O / ml On the other hand, the result of measuring the dissolved oxygen concentration of the same sample using an oxygen electrode was 0. It was 0.41 μmol O / ml, which was almost the same value.

Example 3 4-fold in 50 mM sodium phosphate buffer (pH 6.0)
Hydroxymandelic acid was dissolved to 50 mM to obtain a substrate solution. 400 μl of this substrate solution is measured with a size of 8 × 50.
Dispensed into a mm sample tube (manufactured by Yazawa Chemical Co., Ltd.). The three sample tubes were numbered 0, 1, and 2, respectively, and laccase (derived from Coriolus versicolor IFO 9791, specific activity 4.8 × 10 ³ U /
mg, protein concentration 2.3 mg / ml) 4 μl, 5 μl,
Add 6 μl of water, and then add water (measurement sample) whose dissolved oxygen concentration is to be measured to sample tubes 1 and 2 respectively.
After adding 00 μl and 200 μl, the three sample tubes were capped and gently mixed by inversion several times. After allowing the enzyme reaction by leaving it at room temperature for 30 minutes, 400 μl of 0.01N hydrochloric acid was added to each sample tube and well stirred to stop the enzyme reaction.

Next, 4-hydroxybenzaldehyde produced by the quantitative oxidation reaction of the substrate consuming dissolved oxygen was quantified. That is, 300 μl of the reaction solution was taken from each of the sample tubes 0, 1, 2 and 0.01N hydrochloric acid was added.
After diluting with 4 ml, 200 μl of a 1N hydrochloric acid saturated solution of 2,4-dinitrophenylhydrazine was added to each, and the mixture was allowed to stand for 6 to 8 minutes, and then 5N sodium hydroxide 10
Color was added by adding 0 μl, the absorbance at 500 nm was measured, and the concentration of 4-hydroxybenzaldehyde in each reaction solution was determined by a calibration curve obtained in the same manner from known concentrations of 4-hydroxybenzaldehyde.
The concentration of hydroxybenzaldehyde was determined, and the volume of each reaction solution was used to generate 4 in sample tubes 0, 1, and 2.
The amount of hydroxybenzaldehyde (in μmol units) was determined.

The amount (mol number) of 4-hydroxybenzaldehyde thus obtained was measured with a sample volume of 0 μl, 1
When plotted on the graph with respect to 00 μl and 200 μl, the three points were on a straight line, and the dissolved oxygen concentration (oxygen atom conversion, μmolO / ml) was calculated from the slope.

The measurement sample is at 30 ° C., 40 ° C. under atmospheric pressure.
Distilled water whose dissolved oxygen concentration was saturated by blowing air at each temperature of 50 ° C. and 60 ° C. was used. Further, distilled water in which nitrogen gas was blown to replace dissolved oxygen with nitrogen was also used as a measurement sample. Table 2 shows the values obtained from the dissolved oxygen concentration obtained by the above measurement method and the literature values (edited by the Chemical Society of Japan, Handbook of Chemistry / Basic (Revised 3rd Edition) II, p158).

[Table 2] As shown in Table 2, the measured values of dissolved oxygen concentration by the method of the present invention showed values that corresponded well with the measured values described in the literature.

Claims (1)

[Claims] 1. Dissolved oxygen in a sample in the presence of an oxidase,
General formula: Produced by reacting with a phenol compound represented by
A method for measuring dissolved oxygen concentration, which comprises quantifying hydroxybenzaldehyde.