JPH07177898A - Highly sensitive determination method for tyramine - Google Patents

Abstract

(57) [Summary] [Structure] Benzylamine transaminase and tyramine oxidase are allowed to act on a sample containing tyramine in the presence of an amino group donor such as L-alanine to form an enzyme cycling reaction, and the enzyme cycling is performed. A highly sensitive quantitative determination method for tyramine, which comprises measuring an amplification reaction product such as hydrogen peroxide that is amplified and produced by a reaction. [Effects] The method for quantifying tyramine according to the present invention is capable of quantifying tyramine with high sensitivity without performing inaccuracy and complicated operation of the conventional paper chromatography method and complicated maintenance of high performance liquid chromatography method. It can be performed. Therefore, it has become possible to carry out the quantification of tyramine as a daily work easily, in a short time, with high sensitivity and accuracy.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a highly sensitive method for quantifying tyramine in a subject, and more particularly to a simple and highly accurate method for quantifying tyramine by an enzyme cycling reaction.

[0002]

2. Description of the Related Art Tyramine, which is present in the living body, is a metabolite of catecholamines and is a physiologically active amine which is important for understanding the physiological state of the living body. Therefore, by measuring tyramine, it is possible to obtain important findings in the study of metabolism and physiological actions of catecholamines.

As a method for quantifying tyramine, conventionally, paper chromatography, thin layer chromatography or high performance liquid chromatography has been generally used. Among these methods, the quantitative method which is considered to have high sensitivity and high reliability is a high performance liquid chromatography method. In this method, tyramine is separated and quantified by exactly the same principle as amino acid analysis. For example, in the case of high performance liquid chromatography that uses an ion exchange resin as a packing material, the tyramine adsorbed on the resin is separated by changing the ionic strength or pH of the developing solution, and the separated tyramine is measured for absorbance by the ninhydrin reaction. Or o-
It is detected and quantified by a method such as fluorescence measurement by reaction with phthalaldehyde.

On the other hand, as a method using a principle other than these chromatographic methods, p-hydroxyphenylacetaldehyde, which shows a specific action on tyramine,
Agricultural Biological Chemistry (Agri) that produces ammonia and hydrogen peroxide
c. Biol. Chem. , 29, 117 (1965) (Aspergillus).
A method for quantifying tyramine by an enzymatic method using a tyramine oxidase derived from niger) can be considered.

[0005]

The high performance liquid chromatography method, which has been generally used as a method for quantifying tyramine, has higher sensitivity and accuracy than the paper chromatography method or thin layer chromatography method. Although it is a method, it is difficult to set the analysis accuracy to 2% or less in terms of simultaneous reproducibility and day reproducibility. Further, an expensive high-performance liquid chromatograph apparatus main body is required, and there are many problems and problems such as maintenance and replacement of the packed column and corrosion around the liquid delivery pump due to high ionic strength of the developing solution.

On the other hand, Agricultural Biological Chemistry (Agric. Biol. Che.
m. ), Vol. 29, p. 117 (1965), a method for quantifying tyramine by an enzymatic method using a tyramine oxidase derived from a bacterium (Aspergillus niger) is expected as a simple and highly accurate quantification method for solving the above problems. However, when the tyramine concentration in the sample was 10 μM or less, the sensitivity was insufficient and there was a problem that measurement was impossible.

[0007]

Means for Solving the Problems The inventors of the present invention have earnestly studied a simple and highly sensitive method for quantifying tyramine in order to solve the problems in the conventional quantification method. As a result, they have found that benzylamine transaminase, which is a novel enzyme found by the present inventors, also exhibits activity against tyramine. Further, it was found that tyramine in a sample can be quantified with high sensitivity by forming an enzyme cycle by combining this benzylamine transaminase and tyramine oxidase, and completed the present invention.

That is, the present invention is characterized in that an benzylamine transaminase, an amino group donor, and a tyramine oxidase are allowed to act on an analyte to perform an amplification reaction by an enzyme cycling method, and the resulting amplification reaction product is quantified. It is a highly sensitive method for quantifying tyramine.

The reaction formula of the enzyme cycling in the present invention is shown in the following formula.

[0010]

[Chemical 1]

The reaction principle of enzyme cycling will be described according to the above equation. Tyramine in the test substance is first converted into p-hydroxyphenylacetaldehyde by Tyramine oxidase, and at the same time hydrogen peroxide and ammonia are produced. Then, the converted p-hydroxyphenylacetaldehyde is converted to benzylamine transaminase in the presence of an amino group donor.
It is converted to the original tyramine by the catalytic action of (inase), and at the same time, α-keto acid is produced. By repeating this series of reactions, hydrogen peroxide, ammonia, and α-keto acid are amplified, produced and accumulated. By quantifying at least one of these amplification reaction products, it becomes possible to quantify the amount of tyramine in the subject. In the above reaction formula, L-alanine is used as the amino group donor, pyruvic acid is produced as the α-keto acid, and hydrogen peroxide that is amplified and accumulated is converted into 4-aminoantipyrine (abbreviation: 4-AA). / N-Ethyl-N-(-2-hydroxy-3-sulfopropyl) -m-toluidine sodium (abbreviation: TOOS) / reactions detected by dye formation by the peroxidase method are shown.

The term "analyte" as used in the present invention refers to a liquid such as urine, blood, serum, plasma, culture, culture solution, or intracellular solution containing tyramine, which is a quantitative object, or an extract thereof.

The amino group donor in the present invention is determined by the substrate specificity of the benzylamine transaminase used and is not particularly limited as long as it serves as a substrate for the enzyme. Specific examples of the amino group donor include L-alanine, L-glutamic acid and glycine.

The benzylamine transaminase used in the present invention acts on 1 mol of tyramine and 1 mol of α-keto acid to produce 1 mol of p-hydroxyphenylacetaldehyde and 1 mol of an amino group donor. And, as a reverse reaction thereof, acts on 1 mol of p-hydroxyphenylacetaldehyde and 1 mol of an amino group donor to produce 1 mol of tyramine and 1 mol of α-keto acid. There is no particular limitation, and enzymes having such properties can be used without limitation.

In the present invention, the reverse reaction of benzylamine transaminase is utilized to form an enzyme cycle for use in quantification.

The action of this benzylamine transaminase is shown in the following formula.

[0017]

[Chemical 2]

As an example of such an enzyme, the genus Enterobacter, Bacillus (B)
genus acillus, Agrobacterium
genus Lactobacillus, Corynebacterium (Cor
genus ynebacterium, Comonas
genus monas, Pseudomona
genus As, Brevibacterium
Rium), Streptomyces (Streptomy)
genus ces, Actinomyces
s) genus, Spirillospora
a) genus, Penicillium genus,
Or Neocosmospor
a) Benzylamine transaminase of microbial origin derived from the genus or the like.

The benzylamine transaminase used in the present invention has a strong effect on tyramine, resulting in β-
Alanine, putrescine, and other amines and polyamines that are considered to exist in the living body are preferably used because they do not easily act on them. Examples of the benzylamine transaminase having such advantageous properties for the determination of tyramine in terms of substrate specificity include, for example, Bacillus brevis B-175 (Baci) described in Japanese Patent Application No. 4-327827.
llus brevis B-175, Microbiology Research Institute
3312) -derived benzylamine transaminase.

The tyramine oxidase used in the present invention is an enzyme which catalyzes the reaction of oxidizing 1 mol of tyramine to produce 1 mol of p-hydroxyphenylacetaldehyde, 1 mol of hydrogen peroxide and 1 mol of ammonia. There is no particular limitation, and enzymes having such properties can be used without limitation. The tyramine oxidase used in the present invention is preferably an enzyme that strongly acts on tyramine and hardly acts on amines and polyamines such as β-alanine and putrescine that are present in the body. As an example of a tyramine oxidase having a property advantageous in the determination of tyramine in terms of such substrate specificity, Methods in Enzymology (Methods in Enzymology)
y), Vol. 17B, pp. 722-726 (1971), Sarcina lute.
a, current bacterial name: Micrococcus luteus
IFO-12708) -derived tyramine oxidase, Arthrobacter sp.
Tyramine oxidase derived from Asahi Kasei, product name: TYR
Examples include those derived from microorganisms such as AMINE OXIDASE “TOD”).

The amplification reaction product by enzyme cycling with tyramine oxidase and benzylamine transaminase in the present invention is a compound amplified and produced by the above-mentioned enzyme cycling reaction, and includes hydrogen peroxide, ammonia and α-keto acid. Refers to.

The α-keto acid that is amplified and produced by the enzymatic cycling reaction depends on the amino group donor determined by the substrate specificity of the benzylamine transaminase used in the reaction, and the amino group of the amino group donor used is the carbonyl group. It is a compound having a substituted structure. For example, when the amino group donor is L-alanine, pyruvic acid is produced as an α-keto acid, when the former is glycine, the latter is glyoxalic acid, and when the former is L-glutamic acid, the latter is α-ketoglutar. Each acid is produced.

The conditions for causing the benzylamine transaminase and the tyramine oxidase to act on the test substance may be appropriately selected and used from the conventionally known action conditions of the enzyme, but in general, the benzylamine transaminase used It is desirable that the conditions are such that the activity of the tyramine oxidase is maximized by carrying out the reaction near the optimum pH and temperature of the tyramine oxidase.

The reaction liquid volume is usually in the range of 0.1 to 10 ml.

The pH condition is usually 4.0 to 10.5.
The range of 6.5 to 8.0 is preferable, and a buffer is usually used to maintain the pH in these ranges. The type of the buffer solution is not particularly limited, and a known buffer solution such as a phosphate buffer solution, a citrate buffer solution, a Tris hydrochloric acid buffer solution, or a Good's buffer solution can be used. The concentration of these buffers is not particularly limited, but a range of 2 to 500 mM is preferably used, for example.

The temperature condition is generally in the range of 15 to 60 ° C., but in consideration of practical use, it is especially 30 to 4 ° C.
A range of 0 ° C is preferred.

The amounts of benzylamine transaminase and tyramine oxidase used for quantification in the present invention vary depending on the activity of the enzyme, the reaction conditions, the concentration of tyramine to be measured, etc. and cannot be unconditionally limited, but are preferably set. At the given tyramine concentration, the amount of enzyme is sufficient so that the amplification reaction product is amplified within a predetermined time and accumulated enough for detection and quantification. The amount of benzylamine transaminase and the amount of tyramine oxidase are usually used in the range of 0.1 to 100 U / ml, but 1 to 5
A range of 0 U / ml is preferred.

The amount of the amino group donor used in this enzyme cycling reaction is preferably 5 to 200 times the Km value of the benzylamine transaminase used. Specifically, a concentration range of 0.1 to 500 mM is preferable.

In the enzyme cycling reaction according to the present invention, the reaction proceeds sequentially only when the four components of the amino group donor, tyramine, benzylamine transaminase and tyramine oxidase are present at the same time. Therefore, usually this 4
A means for preparing a test solution containing 2 or 3 of the 1 components and, after reaching the conditions set for the reaction, adding the remaining 1 or 2 components to start the cycling reaction and providing for quantitative determination is adopted. The component added at the end is not limited, but it is common to add the enzyme benzylamine transaminase or tyramine oxidase, or both enzymes at the end.

The reaction time of the enzyme cycling reaction when quantifying tyramine varies depending on the reaction conditions, the concentration of the tyramine to be measured, and the like, and cannot be unconditionally limited, but preferably the enzyme cycling reaction is generated under the set conditions. It is desirable that the time is sufficient for amplification of the product and confirmation of the reaction. As such a reaction time, a range of 1 minute to 5 hours is usually adopted, but particularly 2 minutes to 3 hours.
A range of 0 minutes is preferred.

If it is necessary to stop the enzyme cycling reaction, the reaction is stopped by adding a reaction stopping reagent after the scheduled time. As the reaction stopping reagent, for example, silver chloride which is an inhibitor of tyramine oxidase and benzylamine transaminase is used.

In the highly sensitive method for quantifying tyramine according to the present invention, at least one of hydrogen peroxide, ammonia and α-keto acid which are amplification reaction products by the enzyme cycling reaction may be detected and quantified. The amplification reaction products obtained by these enzyme cycles can be quantified by conventionally known methods.

When the amplification reaction product to be quantified is ammonia or α-ketoglutaric acid which is α-keto acid, L
By an enzymatic method using glutamate dehydrogenase and NADH, an enzymatic method using L-lactate dehydrogenase and NADH when α-keto acid is pyruvate, an enzymatic method using oxaloacetate decarboxylase in the case of oxaloacetate It can be quantified.

When the amplification reaction product to be quantified is hydrogen peroxide, 4-aminoantipyrine-
It can be easily quantified by the peroxidase method. By using the chemiluminescence method for the quantification of hydrogen peroxide, it is possible to make the measurement method with higher sensitivity.

In any case of quantification, a dilution series of a tyramine solution having a known concentration is prepared, and benzylamine transaminase and tyramine oxidase are allowed to act on the solution of this dilution series in advance to produce hydrogen peroxide as an amplification reaction product. , One or more of ammonia and α-keto acid are quantified by the above method, and a calibration curve of the amount of tyramine and the amount of amplification reaction product is prepared. Then, the amount of amplification product can be obtained for the subject by the same method, and the amount of tyramine in the subject can be determined from the calibration curve obtained in advance.

Further, when the amount of tyramine in the test substance is about 1 mM or less, the amount of tyramine as a substrate and the rate of production of the amplification reaction product by the enzyme cycling reaction are in a proportional relationship, so that the amplification reaction product The amount of tyramine in the subject can be calculated from the generation rate. That is, a dilution series of a tyramine solution having a known concentration is prepared, and benzylamine transaminase and tyramine oxidase are allowed to act on the solution of the dilution series, and any of hydrogen peroxide, ammonia and α-keto acid which are amplification reaction products. One or more production rates are measured by the above method, and a calibration curve of the amount of tyramine and the amplification reaction product production rate is prepared. Then, the amplification product generation rate can be obtained for the subject by the same method, and the amount of tyramine in the subject can be determined from the previously obtained calibration curve.

[0037]

The highly sensitive method for quantifying tyramine according to the present invention is
To quantify an amplification reaction product, which is tyramine serving as a substrate, forms an enzyme cycle by utilizing the action of benzylamine transaminase and tyramine oxidase in the presence of an amino group donor, and amplifies and produces an amplification product as a result of the enzyme cycling reaction. Is characterized by.

[0038]

The highly sensitive method for quantifying tyramine according to the present invention provides high sensitivity for tyramine without the inaccuracy and complicated operation of the conventional paper chromatography method and the complicated maintenance of the high performance liquid chromatography method. Can be quantified. In addition, as compared with the conventional measurement method using tyramine oxidase, high sensitivity of about 10 to 500 times can be obtained by performing enzyme cycling. Therefore, even if the amount of tyramine in the subject is very small, it can be quantified. As a result, it has become possible to perform tyramine quantification as a daily routine easily and in a short time with high sensitivity and accuracy.

[0039]

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

Production Example 1 1.0% glucose, 1.0% polypeptone, 0.2%
Yeast extract, 0.1% salt, 0.1% benzylamine,
Sterilized by dispensing 1,500 ml of a medium containing 0.02% antifoaming agent (Ainol) and pH 7.0 (120 ° C, 2
Bacillus brevis B-175 (Microtechnology Research Institute No. 13312) was added to a 5,000 ml Erlenmeyer flask (for 0 minutes).
Was inoculated. After shaking culture at 28 ° C. for 72 hours, this culture solution was added to a jar fermenter preliminarily charged with 20 liters of a medium having the same composition as described above and steam-sterilized to carry out main culture. The culture condition is 28
℃, stirring frequency 150 rpm, aeration rate 20 l / min, 7
After culturing for 0 hour, the culture solution was centrifuged to collect bacterial cells. About 150 g (wet cell weight) of the obtained bacterial cells
Suspend in 500 ml of mM phosphate buffer (pH 7.5),
The suspension was crushed by an ultrasonic crusher. The disrupted liquid was centrifuged using a centrifuge to obtain a supernatant liquid. The total activity of benzylamine transaminase in this supernatant was 5,100 units, and the specific activity was 0.14 units / mg-protein.

The supernatant solution was passed through a column (7φ × 40 cm) packed with DEAE-cellulose (trade name: DE-52 manufactured by Whatman), which had been equilibrated with a 20 mM phosphate buffer solution (pH 7.5) in advance, to pass the enzyme. Adsorbed. After washing this column with 5,000 ml of a phosphate buffer (pH 7.5) containing 10 mM ammonium sulfate, a linear concentration gradient (total volume: 10 liters) of a similar phosphate buffer with an ammonium sulfate concentration of 10 mM to 230 mM was applied. The adsorbed protein was eluted and the benzylamine transaminase active fraction was collected. The total activity of benzylamine transaminase in this active fraction was 1,40.
0 unit, specific activity was 1.25 units / mg-protein.

This active fraction was desalted and concentrated using an ultrafiltration device, and then Sephacryl S-400 (previously equilibrated with 20 mM phosphate buffer (pH 7.5) containing 0.2 M ammonium sulfate). It was passed through a column (6.5 φ × 120 cm) packed with Pharmacia Co., and gel filtration was performed to collect the active fraction. The total activity of benzylamine transaminase in this active fraction was 970 units, and the specific activity was 2.1 units / mg-protein.

Using the enzyme preparation prepared by appropriately diluting the obtained purified enzyme with 10 mM phosphate buffer (pH 7.5), the substrate specificity of this enzyme, optimum pH, pH stability, optimum The suitable temperature and temperature stability were investigated.

[Substrate specificity] 0.2 M phosphate buffer (p
H7.5) 0.74 ml, 50 mM pyruvic acid solution 0.1 ml, 5 mM pyridoxal phosphate solution 0.06 ml, and various 100 mM solutions of amines or amino acids serving as amino group donors. 05 ml
0.05 ml (0.05 unit) of the enzyme preparation was added to the reaction solution consisting of and the reaction was carried out at 30 ° C. for 30 minutes. The reaction solution was immersed in a boiling water bath for 30 seconds to inactivate the enzyme, and then the precipitate was removed by centrifugation to obtain a clear transparent solution. This clear permeate was injected into an amino acid analyzer and L-
Alanine was analyzed quantitatively. The strength of the action of benzylamine transaminase on these amino group donors is shown in Table 1 as a relative activity value when the action on benzylamine is 100%.

[0045] [Optimal pH] 0.2 M various buffers (pH 4.5 to 6.
5: Citrate buffer; pH 6.0-8.0: Phosphate buffer; pH 7.5-9.0: Tris-HCl buffer; pH
9.0 to 11.5: glycine-caustic soda buffer), 0.5 ml of a 5 mM pyridoxal phosphate solution.
1 ml, 0.1 ml of 50 mM pyruvate solution, 10
0.1 ml (0.05 unit) of the enzyme preparation was added to a reaction solution consisting of 0 ml of 0 mM benzylamine hydrochloric acid solution, and the mixture was reacted at 37 ° C. for 5 minutes, and the enzyme was added based on the increase in absorption of benzaldehyde at 250 nm. The activity was sought. After the above operation, the relative activity value was calculated assuming that the maximum enzyme activity value was 100%, and FIG. 1 was obtained. From FIG. 1, it can be seen that the optimum pH of this enzyme is in the range of 7.5 to 8.5.

[PH stability] 0.2M buffer solutions (pH
4.5-6.5: citrate buffer; pH 6.5-8.
0: phosphate buffer; pH 7.5-9.0: Tris-hydrochloric acid buffer; pH 8.0-10.5: glycine-caustic soda buffer) 0.95 ml of 0.05 ml of enzyme preparation (0.
4 units) and left at 60 ° C for 60 minutes,
Add 0.025 ml of each solution to 0.2M phosphate buffer (pH
8.0) 0.5 ml, 5 mM pyridoxal phosphate solution 0.1 ml, 50 mM pyruvic acid solution 0.1 ml, 10
The enzyme activity was determined from the increase in the absorption of benzaldehyde at 250 nm, which was dispensed and mixed in an activity measurement solution consisting of 0.05 ml of 0 mM benzylamine hydrochloric acid solution and allowed to react at 37 ° C. for 5 minutes. Maximum enzyme activity value is 100%
The relative activity value was calculated to obtain FIG. As is clear from FIG. 2, this enzyme is stable in the pH range of 5.5 to 8.5.

[Optimum temperature] 0.2 M phosphate buffer (pH
8.0) 0.5 ml, 5 mM pyridoxal phosphate solution 0.1 ml, 50 mM pyruvic acid solution 0.1 ml, 10
0.1 ml (0.05 unit) was added to an activity measurement solution consisting of 0.05 ml of 0 mM benzylamine hydrochloric acid solution, and 25, 30, 35, 37, 40, 45, 50, 5 was added.
The enzyme activity was determined from the increase in absorption of benzaldehyde at 250 nm under the conditions of reaction at 5, 60, 65, 70, 75 and 80 ° C. for 5 minutes. The relative activity value was calculated with the maximum enzyme activity value as 100%, and FIG. 3 was obtained. From FIG. 3, it can be seen that the optimum temperature of this enzyme is 50 ° C.

[Temperature Stability] 10 mM phosphate buffer (p
0.2 ml (0.04 unit) of enzyme preparation diluted with H7.5) was added to 25, 30, 35, 40, 45, 50, 5
It processed at each temperature of 5,60,65,70 degreeC for 10 minutes.
0.05 ml of this enzyme solution was added to 0.2M phosphate buffer (p
H8.0) 0.5 ml, 5 mM pyridoxal phosphate solution 0.1 ml, 50 mM pyruvic acid solution 0.1 ml, 1
The enzyme activity was determined from the increase in absorption of benzaldehyde at 250 nm, which was dispensed and mixed in an activity measurement solution consisting of 0.05 ml of a 00 mM benzylamine hydrochloric acid solution and allowed to react at 37 ° C. for 5 minutes. Maximum enzyme activity value is 100%
The relative activity value was calculated to obtain FIG. As is clear from FIG. 4, this enzyme is stable at temperatures up to 50 ° C.

Example 1 An enzyme preparation prepared by diluting the purified benzylamine transaminase obtained in Production Example 1 with 10 mM phosphate buffer (pH 7.5), tyramine oxidation derived from Arthrobacter genus manufactured by Asahi Kasei. Enzyme (Product name: TYRAMIN
E OXIDASE “TOD”) and 1.0 μmole / L of tyramine solution as a test substance were used for enzyme cycling to show the relationship between the amount of hydrogen peroxide, which is one of the amplification reaction products, and the reaction time. Examined.

0.5 ml of a 1.0 μmole / L tyramine-hydrochloric acid solution used as a test substance in a cuvette having an optical path width of 1 cm, 200 mM L-alanine, 0.3 mM pyridoxal phosphate, 1 mM N-ethyl-N- ( -2
-Hydroxy-3-sulfopropyl) -m-toluidine sodium, 1 mM 4-aminoantipyrine, and 100 U phosphate buffer (pH 7.5) containing 10 U / ml peroxidase were added in 0.5 ml aliquots and mixed, and then 30
It was heated at 0 ° C for 2 minutes. After heating, 100 μl of the enzyme solution containing 2 U / ml of benzylamine transaminase and 0.5 U / ml of tyramine oxidase obtained in Production Example 1 was added to this cuvette and mixed, and reacted at 30 ° C. The absorbance at 555 nm corresponding to the increased amount of hydrogen peroxide was measured, and the relationship between the reaction time and the absorbance was examined. Results are shown in FIG. As a comparative example, when the enzyme cycling was not performed, the absorbance was measured under the same conditions and operations as in Example 1 except that benzylamine transaminase was not added. The results of this comparative example (displayed as -benzylamine transaminase) are also shown in FIG.

From FIG. 5, it can be seen that the method for carrying out the enzyme cycling according to the present invention can detect tyramine with high sensitivity as compared with the case where benzylamine transaminase is not added.

Example 2 An enzyme preparation prepared by diluting the purified benzylamine transaminase obtained in Production Example 1 with a 10 mM phosphate buffer solution (pH 7.5) and tyramine derived from the genus Arthrobacter manufactured by Asahi Kasei Corporation. Oxidase (Product name: TYRAMI
NE OXIDASE “TOD”) was used to prepare a calibration curve showing the correlation between the tyramine concentration and the production amount of hydrogen peroxide, which is one of the amplification reaction products, by the following method.

0,20 in a cuvette with an optical path width of 1 cm
0,400,600,800,1000,1200nm
0.5 ml of each tyramine-hydrochloric acid solution having ole / L concentration, and 200 mM L-alanine, 0.3 mM pyridoxal phosphate, 1 mM N-ethyl-N-(-2-hydroxy-3-sulfopropyl) -m -Toluidine sodium, 1 mM 4-aminoantipyrine, and 10 U / m
50 mM phosphate buffer (p
After adding 0.5 ml of H7.5) and mixing, the mixture was heated at 30 ° C. for 2 minutes. To each of these cuvettes, 100 μl of an enzyme solution containing 2 U / ml of benzylamine transaminase and 0.5 U / ml of tyramine oxidase obtained in Production Example 1 was added and mixed, and reacted at 30 ° C. for 20 minutes, The absorbance at 555 nm corresponding to the increased amount of hydrogen peroxide was measured to obtain a calibration curve showing the relationship between the tyramine concentration and the absorbance (Fig. 6). As a comparative example, when the enzyme cycling was not performed, the absorbance was measured under the same conditions and operations as in Example 1 except that benzylamine transaminase was not added. The results of this comparative example (displayed as -benzylamine transaminase) are also shown in FIG.

From FIG. 6, it can be seen that the calibration curve for tyramine has good linearity, and the method of the present invention is a highly accurate quantitative method capable of detecting tyramine with high sensitivity.

[Brief description of drawings]

FIG. 1 is a diagram showing a pH activity curve of benzylamine transaminase of the present invention.

FIG. 2 is a diagram showing pH stability of the enzyme.

FIG. 3 is a view showing a temperature activity curve of the same enzyme.

FIG. 4 is a view showing temperature stability of the enzyme.

FIG. 5 shows a reaction curve when enzyme cycling was performed according to the present invention and when enzyme cycling was not performed (indicated as -benzylamine transaminase).

FIG. 6 shows a calibration curve for quantifying tyramine which was subjected to enzyme cycling according to the present invention, and a calibration curve for quantifying tyramine when enzyme cycling was not performed as a comparative example.

Claims (1)

[Claims] 1. A method for increasing the amount of tyramine characterized in that a benzylamine transaminase, an amino group donor, and a tyramine oxidase are allowed to act on an analyte to perform an amplification reaction by an enzyme cycling method, and the resulting amplification reaction product is quantified. Sensitivity quantification method.