JPH0630782A - Method for enzymatic reaction - Google Patents

Abstract

PURPOSE:To simply carry out enzymatic reaction by using city water subjected to at least one residual chlorine eliminating treatment selected from boiling treatment, aeration treatment and addition of thiosulfate. CONSTITUTION:In a method of enzymatic reaction utilizing an isolated or immobilized oxidation-reduction enzyme, a solution prepared by using city water subjected to at least one residual chlorine eliminating treatment selected from boiling treatment, aeration treatment and addition of thiosulfate is used as an aqueous solution to be brought into contact with the enzyme. The aqueous solution is used as a mobile phase in measuring the concentration of a substrate of a flow method and sent to a measuring cell 5 attached to an enzyme electrode 7 by an injector 3. A voltage higher than a reference electrode 8 is impressed to a potensiostate 11 and an active substance of electrode formed or consumed by the enzymatic reaction is electrochemically detected and subjected to data treatment by a computer 14. The enzymatic reaction is simply carried out in this way.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an enzymatic reaction method utilizing at least one oxidoreductase which is free or immobilized, and particularly to an enzymatic reaction method which can be carried out in simple steps.

[0002]

2. Description of the Related Art Enzymes are catalysts that exist for many reactions carried out by living organisms and smoothly proceed under mild conditions in which the living organisms can survive. Therefore, it is easy to proceed only the desired reaction under normal temperature and normal pressure, and the substrate specificity and reaction specificity of the enzyme are widely used in various fields such as production and analysis.

For example, it is applied as a substance-producing catalyst in the production of invert sugar and starch syrup in the field of food production, and is often used as a catalyst for the production of useful substances in the field of drug production. Further, in fields such as food analysis and medical analysis, it is widely used as an analysis catalyst for converting a substance to be measured into a substance to be detected by a detector.

The expansion of the range of utilization of these is because the reaction proceeds under mild conditions in which the enzymatic reaction has a higher reaction efficiency than the chemical methods conventionally used because of its excellent substrate specificity. This is due to advantages such as excellent energy balance. Furthermore, in recent years, adaptation has been made with the progress of utilization technologies such as screening for enzymes with excellent properties, increasing the amount of enzyme production by genetic recombination, modifying the properties of enzymes by protein engineering, and developing enzyme immobilization methods with better reaction efficiency. The range is expanding further.

There are various types of enzymes used as analytical catalysts. Among them, the EC1 group oxidoreductase is a substance that can be detected physically, chemically or electrochemically when it reacts with a substrate. It is a useful enzyme in many cases with increase and decrease. Such enzymes include glucose oxidase (E
C 1.1.3.4), galactose oxidase (E
C 1.1.3.9), pyranose oxidase (EC
1.1.3.10), alcohol oxidase (EC
1.1.13), aldehyde oxidase (EC
1.2.3.1), xanthine oxidase (EC
1.2.3.2), pyruvate oxidase (EC
1.2.3.3), laccase (EC 1.10.3.
2), ascorbate oxidase (1.10.3.
3), etc., which are used as analytical catalysts in various measuring kits and various measuring devices.

Furthermore, these enzymes and other enzymes, namely, transferases in the EC2 group, hydrolases in the EC3 group, EC
It is expected that the scope of analysis will become wider and larger by applying it in combination with the removal and addition enzyme of group 4, the isomerase of group EC5, and the synthase of group EC6. The oxidoreductase can be used as a measurement kit sold by each analytical reagent manufacturer, such as a method in which it is used in combination with a coloring reagent as a solution enzyme, and a measurement device sold by an analytical instrument manufacturer. There are methods such as using as an immobilized enzyme in combination with an enzyme reaction detection device, and in general analysis, quantification of products in enzyme experiments of amylase and glucosidases, glucose in foods, sucrose, maltose, maltooligosaccharides, ethanol,
It is used to know the amount of glucose in urine and blood in medical analysis such as the determination of L-lactic acid and sulfite.

[0007] In general, the enzyme is a substance that is used in the expectation of catalytic action, so that the amount of consumption is small even when using a solution-type enzyme, and further, techniques such as immobilization are processed to enable reuse of the enzyme. It is possible to reduce the running cost of the device and the analysis device. As described above, the reaction method using an enzyme has low running cost and is easy to operate, but may be subject to various restrictions because it is a biocatalyst. For example, even if it can be used under mild conditions, the enzyme protein itself may be denatured or the enzyme activity may be lost under conditions such as extremely high temperature, high pressure, low pH, and high pH. Inhibition and deactivation are caused.
In addition, oxidizing and reducing substances contained in trace amounts in the solution,
In some cases, the active center site that controls the activity of the enzyme and the structure of the enzyme protein itself may change.These restrictions are not limited to the free enzyme because the enzyme is a protein, but to a different extent when using immobilized enzymes. This is also a matter that can be applied.

Usually, in order to overcome such problems,
It is essential to use an aqueous reaction solution suitable for the enzyme and to proceed the enzymatic reaction while controlling the solution temperature if necessary. Furthermore, when precision in the rate and stability of the enzyme reaction is required, such as in enzyme analysis, a buffer solution with pH stability is often used as the enzyme reaction solution. The system solution is required to have the property of not adversely affecting the enzyme.

For these reasons, there is a substance in the reaction solution that loses or inhibits the catalytic property of the enzyme, regardless of whether the enzyme is used as a substance production catalyst or an analysis catalyst. It is not allowed to do so, and particularly in the case of enzyme analysis requiring precision, distilled water or, in extreme cases, ultrapure water is used to prepare the reaction aqueous solution.
This is because practical water cannot be put to practical use because there is a very small amount of a substance that deactivates an enzyme or inhibits the activity in water such as tap water. Since distilled water and ultrapure water require a lot of human power and energy for their preparation, the ratio of the cost required for water to the enzyme analysis was large.

[0010]

However, the use of distilled water, ultrapure water, etc. in this way consumes a great deal of human power and energy for water, which requires labor to prepare an enzyme reaction that can be easily carried out. This has been an obstacle to more easily performing the enzyme reaction. In other words, it cannot be said that the conventionally performed method does not fully utilize the advantages of the enzymatic reaction, which should be simply performed.

The present invention relates to an oxidoreductase which is particularly useful as a substance production catalyst and an analysis catalyst, and pays attention to an aqueous solution used in an enzyme reaction system containing at least one enzyme that is free or immobilized, and is particularly simple. It is an object of the present invention to provide an enzymatic reaction method that can be performed in steps.

[0012]

The present invention is a reaction method utilizing free or immobilized enzyme, and at least selected from boiling treatment, aeration treatment and thiosulfate addition treatment as an aqueous medium in contact with the enzyme. This is an enzymatic reaction method that uses tap water that has been subjected to one residual chlorine treatment. The present invention is a reaction method utilizing a free or immobilized enzyme, which is prepared by using tap water subjected to at least one de-residual chlorine treatment selected from boiling treatment, aeration treatment and thiosulfate addition treatment. An enzyme reaction method using the prepared solution as an aqueous solution in contact with an enzyme.

Further, the above-mentioned enzymatic reaction method wherein the residual chlorine removal treatment is carried out by adding thiosulfate to tap water is disclosed. The present invention is an oxidoreductase in which an enzyme is immobilized, an aqueous solution is a mobile phase used for measuring a substrate concentration in a flow system, and an enzyme reaction method is produced by a reaction between a substrate in a sample and oxidoreductase. Disclosed is the above-mentioned enzyme reaction method, which is a flow-type substrate measurement method by electrochemically measuring the consumed electrode active substance.

[0014]

The aqueous solution in contact with the enzyme can be exemplified by a mobile phase in a flow-type measurement, a washing solution for an enzyme immobilized on an enzyme electrode, a reaction solution in a batch-type measurement using an enzyme, and the like. In the present invention, the aqueous solution is a concept including a sample solution in which a sample is simply dissolved in tap water dechlorinated by the method specific to the present invention, and is not necessarily a buffer solution.

Theoretically, it is desirable that the aqueous solution for carrying out the enzymatic reaction does not contain a substance which is not directly functionally involved in the enzymatic reaction, even if the amount is very small. Impurities for enzymatic reactions can often lead to loss of enzyme activity. In the case of an immobilized enzyme, it can be seen that the properties of the immobilized enzyme may be changed to some extent by the effect of immobilization treatment as compared with the free enzyme, but basically, the properties of the free enzyme are reflected, so that the aqueous solution Impurities cause its deactivation. Since this inactivation phenomenon hinders stable utilization of the enzymatic reaction, its production requires careful attention.

For example, in the case of glucose oxidase, which is one of the redox enzymes, 8-hydroxyquinoline, semicarbazide, sodium nitrate and the like are substances that lose their activity. However, this is just an example, and strong acids and strong alkalis that cause denaturation of the protein itself do not need to be waited for, and even a trace amount of a metal ion, an oxidizing substance, or a reducing substance causes deactivation and inhibition.

In order to avoid such problems, conventionally, distilled water or ultrapure water has been used as the water for the reaction solution or the like. However, if distilled water or ultrapure water is used, the labor and cost of preparing the water becomes large, which hinders easy enzymatic reaction. On the other hand, tap water is the most easily available water, and tap water is stable in terms of water quality because the water quality standards are set by the Ordinance of the Ministry of Health and Welfare so that it does not damage life phenomena even if it is drunk. However, when used as a reaction solution of an enzyme reaction system containing an oxidoreductase, there is a problem that the oxidoreductase is inactivated.

This phenomenon can be presumed to be due to the fact that the active center of the oxidoreductase is affected by the presence of trace amounts of residual chlorine present in tap water. This can be inferred from the fact that the enzymatic activity of oxidoreductase such as glucose oxidase is not affected when distilled water obtained by subjecting tap water to the raw material as distilled water is used as water for the reaction solution or the like.

The above-mentioned treatment such as distillation is one of the simple means for removing the influence of residual chlorine, but it consumes a lot of energy in order to partially vaporize water and recover water vapor. There is. Therefore, if the trace amount of residual chlorine contained in tap water is converted into a substance harmless to the enzyme, or if it is removed, it becomes possible to produce a reaction solution without consuming the energy required for distillation. A means for converting residual chlorine into a substance harmless to the enzyme, or a method for simply removing it is required.

A means that seems to be effective is possible, for example, by adding a compound that reacts with a trace amount of an oxidizing substance or a reducing substance contained in tap water. Since the reaction product of 1 has no adverse effect on the enzyme activity and the surplus of the added compound does not have any adverse effect on the enzyme, its selection is generally carried out without difficulty. The current situation was that they did not.

Therefore, in the present invention, the water quality is stable,
Using easily available tap water as water, an enzyme reaction solution and the like are produced from an aqueous solution that has been subjected to a residual chlorine treatment that can be easily performed. Since the tap water used is water that meets the water quality standards, it can be used without pretreatment such as filtration. Of course, any pretreatment does not hinder the practice of the present invention.

As means for removing residual chlorine, any one of boiling of tap water, aeration of tap water, addition of thiosulfate to tap water, or a combination thereof is performed. When tap water boiling is applied as a means for removing residual chlorine, it is 3 to 30.
The boiling state may be maintained for about 5 minutes, usually about 5 minutes. Usually, it is sufficient to prepare a solution for enzyme reaction such as a buffer solution using tap water that has been boiled and allowed to cool, but if it does not affect additives such as a buffer agent, add each component, after preparation Boiling
It is also possible to allow it to cool.

When tap water is aerated as a means for removing residual chlorine, it is generally aerated because it depends on factors such as the total volume of tap water, temperature, gas flow rate, and contact area between gas and tap water. The time is not fixed, but the method usually applied to the replacement of the dissolved gas is sufficient. Considering that the purpose of the gas used is to replace residual chlorine, chlorine gas is not suitable. In addition, substances that may react with the components of the enzyme reaction solution when dissolved, such as carbon dioxide gas, are not preferable. Therefore, the gas that can be preferably used is nitrogen gas, oxygen gas, air and the like, and air is more preferably used from the viewpoint of cost.

When the addition of thiosulfate to tap water is applied to the means for removing residual chlorine, the thiosulfate that can be added is not limited as long as it is a water-soluble salt, but sodium thiosulfate and thiosulfate can be used. Potassium and the like are preferably used.
The excess thiosulfate does not adversely affect the oxidoreductase, but considering the ionic strength of the enzyme reaction solution and the like, the addition concentration is 0.01 to 50 p. p. m. The range is preferably about 0.1 to 1 p. p.
m. It is a range of degrees. The added thiosulfate reduces chlorine in tap water and converts it into a substance that does not adversely affect the activity of oxidoreductase.

The enzyme reaction solution prepared by using tap water that has been subjected to dechlorination treatment by the production method of the present invention is used for precise enzyme reaction measurement such as enzyme analysis without impairing the activity of oxidoreductase. It is possible. When an aqueous solution is used as the mobile phase for the flow measurement, tap water is subjected to at least one boiling treatment, aeration treatment, or thiosulfate addition treatment to perform phosphoric acid-NaOH or Tris-
Prepare a buffer such as HCl that is usually used for enzyme reaction. Also, when using as a reaction solution in batch type measurement, a buffer solution is similarly prepared by using dechlorinated tap water.

When an aqueous solution is used as a cleaning solution for enzyme electrodes and the like, tap water which has been subjected to at least one dechlorination treatment among boiling treatment, aeration treatment and thiosulfate addition treatment is used as it is, or If necessary, prepare it in a buffer and use it. It can also be used as a cleaning solution for measuring devices, a solution for diluting a sample, and the like. When a free enzyme is used, an aqueous solution can be used as a solution for diluting the enzyme, a storage solution, or the like.

As the enzymatic reaction method, measurement using the above-mentioned oxidoreductase, for example, measurement of glucose using glucose oxidase, measurement of ethanol using alcohol oxidase, L-use of L-lactate oxidase
Examples include measurement of lactic acid. In addition, oxidoreductase and E
C2 group transferase, EC3 group hydrolase, EC4 group removal addition enzyme, EC5 group isomerase, and EC6 group synthase is there. As an example, invertase (EC 3.2.1.26) and mutarotase (EC
5.1.3.3) and glucose oxidase in combination to measure sucrose, L-lactate dehydrogenase (EC
1.1.1.27) -Pyruvate measurement in combination with L-lactate oxidase can be mentioned.

[0028]

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 Glucose oxidase was used as an oxidoreductase. (1) Preparation of solution for enzyme reaction Sodium thiosulfate (molecular weight 158.
0.632 mg was added to 0.632 p. p. m.
The concentration of Using this tap water, a 100 mM phosphate buffer containing 50 mM potassium chloride and 1 mM sodium azide was prepared, and the pH was adjusted to 6.0. (2) Method for making enzyme electrode A 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 (hereinafter abbreviated as SCE) as a reference electrode, electrolytic treatment is performed in 0.1 M sulfuric acid at +1.4 V for 5 minutes.

After that, the platinum wire was thoroughly washed with water, dried at 40 ° C. for 10 minutes, dipped in an anhydrous toluene solution of 10% γ-aminopropyltriethoxysilane for 1 hour, and then washed. <Formation of Enzyme-Containing Membrane> An enzyme system responsive to glucose was immobilized on the aminosilanized platinum wire as follows. Glucose oxidase (manufactured by Sigma, type I) in 100 mM sodium phosphate buffer (pH 6)
I) 5 mg / ml, bovine serum albumin (Sigma, F
ration V) 5 mg / ml and 5 μl of a solution containing 0.2% of glutaraldehyde are added dropwise, and 1
Immobilization was performed by treating for 5 minutes. (3) Measuring apparatus A flow type measuring apparatus having an enzyme electrode arranged as shown in FIG. 1 was used.

This flow-type measuring device is equipped with an injector 3 for high performance liquid chromatography (712 manufactured by Rheodyne Co., Ltd.).
5 type) and the measuring cell 5 to which the enzyme electrode 7 produced by the above-described procedure is attached are connected by a fluororesin pipe 4 having an inner diameter of 0.5 mm and a length of 1.0 m. Inner volume 40 μl
An enzyme electrode 7 and an Ag / AgCl reference electrode 8 are arranged to face each other in the measuring cell 5 of (1) via a buffer line, and a pipe made of stainless steel attached as a counter electrode 9 is connected to the measuring cell 5. ing. These are installed inside the constant temperature bath 10, and the temperature inside the bath is maintained at 37 ° C ± 0.2 ° C. A voltage of +0.6 V is applied to the enzyme electrode 7 by the potentiostat 11 with respect to the Ag / AgCl reference electrode so that hydrogen peroxide generated at the electrode is finally detected as a target for electrochemical measurement. did. The detection signal was transferred to the computer 14 via the A / D converter 12 and the communication cable 13 for data processing.

For sending the buffer solution from the buffer solution reservoir 1, the solution sending pump 2 is controlled by a computer.
It is set so that liquid is delivered at a flow rate of 0 ml / min. The buffer is the enzyme reaction solution according to the present invention. The buffer solution after the measurement is captured by the waste liquid reservoir 6. (4) Measuring method After the temperature of the constant temperature bath reached equilibrium, add 5 parts of glucose standard solution.
μl was injected.

After confirming that a proportional relationship was established between the detected value and the concentration up to a concentration of 40 mM, a glucose solution having a concentration of 10 mM was injected every hour. (5) Results The measurement results are shown in Table 1 and FIG. The unit of the output value of the glucose electrode is μA. The unit of the elapsed time of feeding the solution for enzyme reaction is time.

[0034]

[Table 1]

No decrease in glucose oxidase activity was observed even after 5 hours from the start of feeding the enzyme reaction solution.

Comparative Example 1 (1) Preparation of Enzyme Reaction Solution Using untreated tap water, 50 mM potassium chloride, 1 m
A 100 mM phosphate buffer containing M sodium azide was prepared and the pH was adjusted to 6.0. (2) Method for producing enzyme electrode Same as in Example 1. (3) Measuring device Same as in Example 1. (4) Measurement method Same as in Example 1. (5) Results The measurement results are shown in Table 2 and FIG. The unit of the output value of the glucose electrode is μA. The unit of the elapsed time of feeding the solution for enzyme reaction is time.

[0037]

[Table 2]

After 5 hours from the start of feeding the enzyme reaction solution, the activity of glucose oxidase was about 8% of the initial level.
It fell to 9%.

Example 2 As a means for removing residual chlorine, tap water was boiled. (1) Preparation of Enzyme Reaction Solution 1 liter of tap water was placed in a glass beaker and heated with a gas burner. With the beaker open, the mixture was boiled for 15 minutes after the start of boiling and allowed to cool to room temperature (27 ° C). Using this tap water, a 100 mM phosphate buffer containing 50 mM potassium chloride and 1 mM sodium azide was prepared, and the pH was adjusted to 6.0. (2) Method for producing enzyme electrode Same as in Example 1. (3) Measuring device Same as in Example 1. (4) Measurement method Same as in Example 1. (5) Results The measurement results are shown in Table 3 and FIG. The unit of the output value of the glucose electrode is μA. The unit of the elapsed time of feeding the solution for enzyme reaction is time.

[0040]

[Table 3]

No decrease in glucose oxidase activity was observed even after 5 hours from the start of feeding the enzyme reaction solution.

[0042]

Industrial Applicability According to the present invention, it has become possible to produce an enzyme reaction solution for oxidoreductase and the like in a simple process.
In Examples 1 and 2, there is no change in the output value of the glucose electrode even if the measurement of 8 hours per day is performed for 7 days, and the measurement is performed in the same manner as when the enzyme reaction solution was prepared using distilled water or the like. I was able to do it.

On the other hand, in Comparative Example 1, when the measurement for 8 hours per day was performed for 3 days, the output value of the glucose electrode almost disappeared and the measurement could not be performed.

[Brief description of drawings]

FIG. 1 is an example of a configuration diagram of a glucose measuring device used in Examples 1, 2 and Comparative Example 1.

FIG. 2 shows the measurement results in Example 1. The vertical axis represents the output value of the glucose electrode, and the unit is μA. The horizontal axis represents the elapsed time after feeding the enzyme reaction solution, and the unit is time.

FIG. 3 illustrates the measurement results in Comparative Example 1. The vertical axis represents the output value of the glucose electrode, and the unit is μA. The horizontal axis represents the elapsed time after feeding the enzyme reaction solution, and the unit is time.

FIG. 4 illustrates the measurement results in Example 2. The vertical axis represents the output value of the glucose electrode, and the unit is μA. The horizontal axis represents the elapsed time after feeding the enzyme reaction solution, and the unit is time.

[Explanation of symbols]

 1 Buffer Reservoir 2 Liquid Feed Pump 3 Injector 4 Piping 5 Measurement Cell 6 Waste Liquid Reservoir 7 Enzyme Electrode 8 Ag / AgCl Reference Electrode 9 Counter Electrode 10 Thermostat 11 Potentiostat 12 A / D Converter 13 Communication Cable 14 Computer 15 Printer

Claims (3)

[Claims] 1. A reaction method using a free or immobilized enzyme, which comprises using tap water subjected to at least one de-residual chlorine treatment selected from boiling treatment, aeration treatment and thiosulfate addition treatment. An enzyme reaction method using the prepared solution as an aqueous solution in contact with an enzyme. 2. The enzymatic reaction method according to claim 1, wherein the residual chlorine removal treatment is performed by adding thiosulfate to tap water. 3. An enzyme is an immobilized oxidoreductase, an aqueous solution is a mobile phase used for flow-type substrate concentration measurement, and an enzyme reaction method is generated by a reaction between a substrate in a sample and oxidoreductase. The enzyme reaction method according to claim 1, which is a flow-type substrate measuring method by electrochemically measuring the consumed electrode active substance.