JPH11253198A - Enzymatic analysis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of enzymatic analysis enabling a wide range of substrate concentrations to be determined. SOLUTION: This method of enzymatic analysis comprises using a plurality of enzymes which recognize the identical substrate and differ in Km value from one another. Thereby, the above enzymes serve as a biosensor for glucose.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a sugar using an enzyme,
The present invention relates to a method for quantifying chemical substances such as alcohols, organic acids, and amino acids. More specifically, it is used in fields such as clinical tests and food analysis.

[0002]

BACKGROUND OF THE INVENTION Sugars, alcohols, organic acids,
BACKGROUND ART A method for quantifying a chemical substance such as an amino acid, a so-called enzyme analysis method, pays attention to the excellent substrate specificity and reaction specificity of an enzyme, and is applied to clinical tests, food analysis, and the like. In the enzyme analysis method, usually, an enzyme that performs molecular recognition for recognizing one specific compound, that is, one enzyme (first enzyme) that recognizes a target compound is determined. For example, in the measurement of glucose, glucose oxidase that specifically recognizes glucose and oxidizes it, or glucokinase that specifically recognizes glucose and phosphorylates it is used as the first enzyme. In the case of using glucose oxidase, since the determination of the amount of generated hydrogen peroxide is made to be a Merck mark, the analysis kit is further prepared by combining peroxidase with a chromogenic dye as an enzyme using hydrogen peroxide as a substrate. To construct. In the latter case, glucose, the reaction product
A glucose analysis kit is prepared by combining with glucose-6-phosphate dehydrogenase which specifically recognizes and oxidizes hexaphosphate. In this way, the concentration of the target compound is measured by directly or indirectly measuring the compounds produced and consumed in the measurement.In any case, one kind of enzyme that recognizes the target compound is used. Enzymes have been used. This is also used for the principle of an enzyme sensor that measures the concentration of a compound using an enzyme as a molecular recognition element, and again, only one enzyme recognizes a target compound. Therefore, factors that limit the measurement limit and the like in the analysis method using these enzymes are mainly governed by enzymatic properties such as affinity of the first enzyme with the substrate.

[0003]

SUMMARY OF THE INVENTION In an enzyme analysis method, the affinity of a first enzyme for a substrate is a major factor that governs a measurement system. In other words, in the enzyme analysis method, it is desirable that a linear relationship be established between the observed value and the concentration of the target substance.However, since the enzyme usually draws a Michaelis Menten-type saturation curve, the substrate concentration range is extremely limited. Only at, a linear relationship holds. Therefore, when preparing an enzyme analysis kit, based on the Km value of the first enzyme, if the sample is not appropriately prepared, the reliability cannot be obtained in the measurement. Therefore, in order to cover a wide range of substrate concentrations, pretreatment such as pre-dilution of the sample must be performed.

[0004]

OBJECTS OF THE INVENTION The present invention seeks to overcome the disadvantages of the prior art and to provide an enzyme assay having a wide dynamic range. The present invention relates to a sugar using an enzyme,
It relates to a method for quantifying chemical substances such as alcohols, organic acids, and amino acids, and can be applied to extremely wide fields such as clinical tests and food analysis.

[0005]

SUMMARY OF THE INVENTION In consideration of the actual conditions of such an enzyme analysis method, the present inventors have been diligently working to improve the conventional enzyme analysis method and to develop an enzyme analysis method having a wide dynamic range. Was performed. As a result, they found that the use of a plurality of enzymes having different Km values that recognize the same substrate as the first enzyme greatly expanded the quantification range of the target substrate. Specifically, it recognizes the same substrate, and
It has been found that an enzyme analysis kit and an enzyme sensor having a 1000-fold dynamic range can be constructed by preparing an analysis kit for a target compound using two or more enzymes whose Km values differ by about 10 times as the first enzyme.

In order to solve the above-mentioned problems, two or more enzymes that recognize the same substrate and differ in Km value by about 10 times are used as first enzymes.
It may be used as an enzyme. As a combination of such enzymes, two or more natural enzymes that have already been reported, for example, alcohol oxidase and PQQ-dependent alcohol dehydrogenase for measuring alcohol, and cholesterol derived from actinomycetes for measuring cholesterol Glucose oxidase and PQQ-dependent glucose dehydrogenase for the measurement of oxidase and cholesterol oxidase derived from microorganisms, glucose, and malate dehydrogenase derived from porcine heart and malate dehydrogenase derived from microorganisms for quantification of malic acid, glycerol The quantification of NAD + -dependent glycerol dehydrogenase derived from Escherichia coli and NADP + -dependent glycerol dehydrogenase derived from rabbit skeleton include, but are not limited to. Furthermore, as shown in Examples described later, the amino acid sequence of a natural enzyme is modified by a protein engineering technique, and the constructed modified enzymes having different substrate recognition abilities can be combined with each other or in combination with a natural enzyme. Achieved.

[0007] Using these enzymes, it is possible to quantify the substrate based on various known analytical methods. For example, for oxidoreductase, phenazine methosulfate,
These may be quantified by colorimetry using an electron mediator such as 1-methoxyphenazine methosulfate, dichlorophenol indophenol, ferricyanide compound, ferrocene, ferrocene derivative, benzoquinone, nitroblue tetrazolium chloride, and the like.
Alternatively, the reaction product may be quantified using a spectrophotometer, a differential refractometer, a fluorometer, or the like, in combination with an enzyme using the reaction product as a substrate or an enzyme using the reaction product as a substrate, and a coloring dye.

A biosensor having a wide dynamic range can be constructed using these enzymes. The biosensor that can be used in the present invention can be constructed based on a known method. For example, these enzymes are immobilized by an enzyme immobilization method generally used for a gold electrode, a glassy carbon electrode, a platinum electrode, a carbon paste electrode, and the like, for example, an adsorption method, a crosslinking method, a covalent bonding method, and the like. A biosensor may be constructed using it.

In the determination of glucose, two or more kinds of enzymes that specifically recognize glucose and have a Km value that is about 10 times different may be used as the first enzyme. For example, glucose oxidase that oxidizes glucose, glucose dehydrogenase, or glucokinase, which specifically recognizes glucose and phosphorylates it, uses two or more enzymes whose Km values differ by about 10 times. Good. These Km
Two or more enzymes whose values differ about 10 times
Glucose can be quantified based on a known analysis method. For example, when using glucose dehydrogenase, it is possible to construct a glucose analysis method by combining the quantification of a reduced form of the electron acceptor with a known analysis method, for example, a colorimetric quantification method, in combination with a known electron acceptor. it can.

[0010] A sensor can be used for the determination of glucose. For example, when using glucose dehydrogenase, the electron acceptor and glucose dehydrogenase may be dissolved or dispersed in a sample solution, or may be adsorbed to a membrane or the like by a known immobilization method such as an adsorption method, a crosslinking method, or a covalent bonding method. Examples include a type immobilized and mounted on the surface of a glassy carbon electrode or the like, and a type immobilized on a surface of a glassy carbon electrode or the like by a known immobilization method such as an adsorption method, a crosslinking method, or a covalent bonding method. It may be appropriately selected and constructed according to the type of glucose dehydrogenase or electron acceptor. The example illustrated here is an amperometric glucose sensor. According to the sensor, electron transfer accompanying a reaction can be directly detected on the electrode surface. That is, the reduced form of the electron acceptor is electrochemically quantified by using the glucose dehydrogenase as described above. Alternatively, electron transfer accompanying the reaction is directly quantified.

[0011]

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Example 1 Glucose dehydrogenase (PQQGDH) using pyrroloquinoline quinone (PQQ) as a coenzyme as an enzyme that recognizes glucose
Was combined with modified PQQGDH and H775D whose affinity for glucose was modified by a protein engineering technique to prepare a glucose analysis kit having a wide dynamic range. H775D is Escheri
This enzyme is an enzyme in which the histidine residue at position 775 of chia coli membrane-bound PQQGDH has been modified to an aspartic acid residue by site-directed mutagenesis, specifically oxidizes glucose, and has a Km value for glucose of about 20 mM. It is about 20 times that of type PQQGDH (Biotechnol. Lett., 1997 K. Sode, K. Kojima).

The amount of the enzyme was adjusted so that the same activity was obtained when the wild-type enzyme was reacted at a glucose concentration of 20 mM and the H775D modified enzyme was reacted at a glucose concentration of 500 mM. The glucose concentration was measured in the presence of 1 mM PMS, 0.1 mM DCIP, pH 7.0, 10 mM potassium phosphate buffer, using the bleaching of DCIP at 600 nm as an index. The result is shown in FIG. With each enzyme alone, a glucose concentration equivalent to about 1/10 to 10 times the Km value can be measured, but the measured value is unreliable in a range higher than that. On the other hand, in the analysis kit prepared by mixing the wild-type enzyme and H775D, measurement was possible in a glucose concentration range of 1000 times from glucose concentration of about 0.1 mM to 100 mM.

Example 2 Glucose dehydrogenase (PQQGDH) using pyrroloquinoline quinone (PQQ) as a coenzyme as an enzyme that recognizes glucose
By combining this with modified PQQGDH and H775D whose affinity for glucose was modified by a protein engineering technique, an amperometric biosensor for glucose quantification having a wide dynamic range was prepared.

The enzyme concentration was adjusted so that when the wild-type enzyme was reacted at a glucose concentration of 20 mM and the H775D modified enzyme was reacted at a glucose concentration of 500 mM, the activity value was 5 units / ml. 100mg of carbon paste and TSK g as basic carrier
el DEAE Toyopearl 650M (manufactured by Tosoh Corporation) 10mg mixed and filled with 3mm inner diameter carbon paste electrode (BAS
(Manufactured by Co., Ltd.) was immersed in 1 ml of the prepared enzyme solution for 30 seconds to immobilize the enzyme on the electrode. After that, this carbon paste electrode
The surfactant Triton X-100 was removed from the surface of the carbon paste electrode by immersing in 10 mM potassium phosphate buffer pH 7.0 and stirring. By the above operation, a glucose dehydrogenase-immobilized electrode was prepared.

The glucose dehydrogenase-immobilized electrode prepared by the above method was used as a working electrode, silver / silver chloride was used as a reference electrode, and platinum was used as a counter electrode. Methoxyphenazine methosulfate was used as the electron acceptor, and the working electrode, the reference electrode, and the counter electrode were immersed in 10 ml of 10 mM potassium phosphate buffer pH 7.0 containing 1 mM methoxyphenazine methosulfate. For quantification of glucose, a potential of +10 mV vs Ag / AgCl was applied between the working electrode and the counter electrode, and a current value generated by the addition of glucose was measured with an ammeter and recorded on a recorder.

FIG. 2 shows the results. Each enzyme alone is 0.1 mM to 1 mM for the wild-type enzyme and 1 for the H775D modified enzyme.
Measurement of glucose from mM to 10 mM can be performed, but the measurement value is unreliable in the range higher than that. 0.1 mM on the enzyme electrode immobilized with both wild-type enzyme and H775D mutant enzyme
Direct measurement of about 10 mM glucose was possible.

[0018]

Since the present invention is constructed and functions as described above, the target substance can be quantified in a wide dynamic range. That is, reliable measurement can be performed without any operation of sample preparation such as diluting and concentrating the sample. The enzyme analysis method of the present invention can be used in an extremely wide field.

[Brief description of the drawings]

FIG. 1 is a diagram showing a calibration curve in one embodiment of the present invention.

FIG. 2 is a diagram showing a calibration curve in another example of the present invention.

Claims (4)

[Claims] 1. An enzyme analysis method comprising two or more enzymes which recognize the same substrate and have different Km values as main components. 2. A biosensor constructed according to claim 1. 3. The enzyme analysis method according to claim 1, wherein the measurement target is glucose. 4. The biosensor according to claim 2, wherein the measurement target is glucose.