JPS636457A - Enzyme activity test method for immobilized enzyme membrane - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] [Technical field to which the invention pertains] The present invention relates to a method for testing the enzyme activity of an immobilized enzyme membrane used when quantifying the concentration of 29 substances in a solution using an enzyme electrode method. The enzyme activity of the immobilized enzyme membrane can be improved without directly attaching the immobilized enzyme membrane to the electrode, thereby preventing wrinkles, peeling scratches, etc. from occurring on the membrane, and thus ensuring the properties when attached to the electrode again. Concerning test methods.

[Prior art and its problems]

The enzyme electrode method is currently widely used in the clinical, food, and environmental fields as a method for quickly, easily, and highly accurately quantifying analyte substances in specimens.

However, it is usually very difficult to immobilize an enzyme so that the enzyme activity remains constant without variation during the process of forming an immobilized enzyme membrane. Therefore, inspection of enzyme activity after membrane formation is extremely important for quality control of immobilized enzyme membranes.

Conventionally, this type of enzyme activity evaluation method uses an immobilized enzyme membrane 2 held on an O-ring 22 as shown in FIG. 2(A).
1 is attached to the electrode surface of a solid electrode consisting of an indicator electrode 23 and a counter electrode 24 as shown in FIG. 2(B), and inserted into the measurement cell 27 shown in FIG. A so-called enzyme electrode method is known in which the output of a substrate solution (sample solution) 25 of concentration is measured. In FIG. 2(C), 26 is a sample liquid flow path. However, in this method, since the immobilized enzyme membrane is attached to the electrode, force is applied to the membrane surface, which tends to cause wrinkles and peeling scratches when the membrane is separated, and it is difficult to guarantee the characteristics when attached to the electrode again. The drawback was that it couldn't be done.

[Purpose of the invention]

The purpose of the present invention is to avoid attaching the immobilized enzyme membrane to be tested directly to the electrode, thereby preventing wrinkles, peeling scratches, etc. from occurring on the membrane, and ensuring the same characteristics when attached to the electrode again. An object of the present invention is to provide a non-destructive method for testing the enzyme activity of an immobilized enzyme membrane, which overcomes the drawbacks of the known techniques.

[Key points of the invention]

In order to achieve the above-mentioned object, according to the present invention, an immobilized enzyme membrane used when quantifying a substrate concentration in a solution by an enzyme electrode method is added to a substrate solution having a predetermined concentration, volume, and temperature while stirring. After a certain period of time, the immobilized enzyme membrane is lifted from the substrate solution, and the electrode-active enzyme reaction product contained in the substrate solution is quantified by electrochemical means to determine the enzyme activity of the immobilized enzyme membrane. It is characterized by evaluating.

That is, the present invention provides an immobilized enzyme membrane with a predetermined concentration, volume,
Enzyme reaction conditions are kept constant by immersion in a substrate solution at a certain temperature, and physical damage to the membrane is prevented, and the delimited enzyme membrane is pulled out of the substrate solution after a certain period of time under the above reaction conditions. The purpose is to evaluate the enzyme activity within the immobilized enzyme membrane by stopping the enzyme reaction and electrochemically quantifying the electrode active substance produced by the enzyme reaction.

In the present invention, the enzyme supported in the immobilized enzyme membrane is an oxidoreductase, and the enzyme reaction product is arsenic peroxide or oxygen.

Further, in the present invention, the electrochemical means is a polarographic method or an enzyme electrode method using an electrode equipped with a selectively permeable membrane that selectively permeates oxygen or hydrogen peroxide.

[Embodiments of the invention]

Rattan Grip - Figure 1 above is a reactor for carrying out the method of the invention. The immobilized enzyme membrane 1 is placed together with a predetermined amount of standard substrate solution 2 into a glass container 3 equipped with a stirrer 5 at the bottom. Furthermore, this glass container 3 is inserted into a container 8 in which a silicone tube 6 for flowing constant temperature water 7 is wound into a coil, and water 4 is filled between these two containers 3.8. . This water 4 is used as a lubricant for rotating the entire glass container 3 by the magnetic stirrer 10.
and serves as a heat transfer medium. The outside of the silicon tube 6 is covered with a heat insulating material 9, thereby preventing heat dissipation of the constant temperature water 7 to the outside.

As a result of experiments using the above-mentioned reactor, the temperature fluctuation of the substrate solution in the glass container was ±0.3°C, and the substrate solution could be sufficiently stirred without physically damaging the immobilized enzyme membrane. I understand.

Tensan ±-1 Using the reactor of Example 1, an alcohol oxidase (AOD) membrane was used as the immobilized enzyme membrane, and 10% as the substrate solution.
1 ml of EtOH-pH 7.0 phosphate buffer, set temperature 3
The reaction was carried out at 0°C. AOD is calculated using formula (1)% formula% (1
), it is an enzyme that oxidizes ethanol to acetaldehyde, consuming oxygen and producing hydrogen peroxide. The hydrogen peroxide concentration generated in the substrate solution was determined by attaching an acetyl rose membrane to the solid electrode shown in Figure 2.
0.64 V was applied to the indicator electrode 23, and 10 μl of the substrate solution in the middle of the reaction was injected into the measurement cell 27, and the value was determined based on the oxidation current value of hydrogen peroxide flowing at that time.

FIG. 3 is a graph showing changes over time in the concentration of hydrogen peroxide produced in the substrate solution. Since the concentration of hydrogen peroxide produced increases linearly with time, it can be seen from FIG. 3 that the enzyme reaction proceeds as a pseudo-order reaction.

Furthermore, glucose oxidase (C
OD) 500■/d as membrane and substrate solution! Glucose-
Experiments were conducted using pH 7.0 phosphate buffer. COD
oxidizes glucose to gluconic acid as shown in formula (2), ie, consumes oxygen and produces hydrogen peroxide similarly to AOD. It was found that the hydrogen peroxide concentration produced increased linearly with time in the range of 0 to 60 minutes, similar to AOD.

From the above results, the enzyme activity within each immobilized enzyme membrane can be evaluated by measuring the concentration of hydrogen peroxide produced over a certain period of time.

In view of the experimental results of Example 2 regarding the five main AOD membranes, the reaction time was 30 minutes, the substrate solution was 200μ2, and multiple AOD membranes were used.
The hydrogen peroxide concentration produced on the membrane was measured. Furthermore, each AOD membrane was attached to an electrode, the output was measured using an enzyme electrode method, and the relationship with the generated hydrogen peroxide concentration was investigated. The results are shown in FIG.

In FIG. 4, the vertical axis is the concentration of hydrogen peroxide produced under the above conditions, and the horizontal axis is the output of 20% ethanol using the enzyme electrode method. However, the output of the enzyme electrode was divided by the output of a 60 ppm hydrogen peroxide solution to correct for fluctuations in the sensitivity of the electrode itself to hydrogen peroxide.

If the concentration of hydrogen peroxide produced is measured from the graph in Figure 4,
The sensor output when attached to the electrode can be estimated. Therefore, it can be seen that the enzyme activity evaluation test of the immobilized enzyme membrane according to the present invention is effective in reducing the variation in the output of enzymes.

Furthermore, in the above-mentioned embodiments, the concentration of hydrogen peroxide produced was used as the chemical species to be measured, but the difference in dissolved oxygen concentration before and after the reaction, that is, the concentration of oxygen consumed in the enzymatic reaction, may also be measured. At that time, the inside of the glass container 3 shown in FIG. 1 was replaced with inert gas,
Must be sealed.

Furthermore, to detect the oxygen concentration, the voltage applied to the indicator electrode 23 is set to 0.
．． A Bolaro type oxygen electrode with a voltage of 60V is used.

〔Effect of the invention〕

According to the present invention described above, the immobilized enzyme membrane is immersed in a substrate solution of a predetermined concentration, volume, and temperature for a certain period of time, and the electrode-active reaction product is quantified by electrochemical means. Enzyme activity can be accurately evaluated without causing physical damage to the enzyme. As a result, not only is there no loss of membranes for evaluating enzyme activity, but it is also possible to perform screening using a complete test.

[Brief explanation of the drawing]

FIG. 1 is a cross-sectional view of a reactor for carrying out the method of the present invention, and FIGS. FIG. 3 is a graph showing the change over time in the concentration of hydrogen peroxide produced, and FIG. 4 is a graph showing the relationship between the concentration of hydrogen peroxide produced and the output of the enzyme electrode. ■, 21: Immobilized enzyme membrane, 2: Substrate solution, 3: Glass container, 5: Stirrer, 6: Silicone tube, 7: Constant temperature water, 10: Magneto stirrer,

Claims (3)

[Claims] (1) An immobilized enzyme membrane used when quantifying the substrate concentration in a solution using the enzyme electrode method is immersed in a substrate solution of a predetermined concentration, volume, and temperature with stirring, and after a certain period of time, the membrane is immersed in a substrate solution. An immobilized enzyme characterized in that the enzyme activity of the immobilized enzyme membrane is evaluated by pulling up the immobilized enzyme membrane and quantifying the electrode-active enzyme reaction product contained in the substrate solution by electrochemical means. Membrane enzyme activity testing method. (2) In the method according to claim 1, the enzyme supported in the immobilized enzyme membrane is an oxidoreductase,
A test method in which the enzymatic reaction product is hydrogen peroxide or oxygen. (3) In the method described in claim 1, the electrochemical means may include a polarographic method or an enzyme electrode method using an electrode equipped with a selectively permeable membrane that selectively permeates oxygen or hydrogen peroxide. Test method used.