JP3155415B2 - Highly sensitive method for determination of benzylamines - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for highly sensitive determination of benzylamines in a test sample, and more particularly to a simple and accurate method for quantitatively determining benzylamines by an enzyme cycling reaction.

[0002]

2. Description of the Related Art Benzylamines present in living bodies and the like are regarded as metabolic derivatives of catecholamines. Therefore, by measuring benzylamines, it is possible to obtain important findings in studying the metabolism and physiological effects of catecholamines.

[0003] As a method for quantifying benzylamines, conventionally, a method using paper chromatography, thin-layer chromatography or high-performance liquid chromatography has been generally used. Among these methods, a high-performance liquid chromatography method is a highly sensitive and reliable quantitative method. In this method, benzylamines are separated and quantified by exactly the same principle as in amino acid analysis. For example,
In the case of high performance liquid chromatography using an ion exchange resin as a packing material, benzylamines adsorbed on the resin are separated by changing the ionic strength or pH of the developing solution, and the separated benzylamines are subjected to a ninhydrin reaction. It is quantified by a method such as absorbance measurement or fluorescence measurement by reaction with o-phthalaldehyde.

[0004] On the other hand, as a method using a principle other than the chromatographic method, agricultural biological chemistry (Agr) which acts on benzylamines to generate benzaldehydes and hydrogen peroxide is used.
ic. Biol. Chem.), vol. 29, p. 117 (1965).
A quantification method of benzylamines by an enzymatic method using an amine oxidase derived from Aspergillus niger is considered.

[0005]

The high-performance liquid chromatography method, which has been generally used as a method for quantifying benzylamines, has a higher sensitivity and accuracy than the paper chromatography method or the thin-layer chromatography method. Although it is a high quantitative method, it is difficult to reduce the analytical accuracy to less than 2% in terms of simultaneous reproducibility and daily reproducibility.
Further, an expensive high-performance liquid chromatography apparatus body is required, and there are many problems and problems such as maintenance / replacement of the packed column and corrosion near the liquid sending pump due to high ionic strength of the developing solution.

On the other hand, Agricultural Biological Chemistry, Vol. 29, p. 117
(1965) The method for quantifying benzylamines by an enzymatic method using an amine oxidase derived from mold (Aspergillus niger) described in (1965) was expected as a quantitative method for solving the above problems, but the enzyme activity of amine oxidase was Was found to be significantly inhibited by anionic substances such as chloride ions, and it was found that this was not a practical method of quantification.

[0007]

Means for Solving the Problems The present inventors have intensively studied a simple and high-precision quantification method for benzylamines in order to solve the problems in the conventional quantification method. As a result, Agricultural Biological Chemistry (Agric.Biol.Chem.), Vol. 29, page 117 (1965) described mold (Aspergillus niger) derived benzylamines quantitative system by amine oxidase derived from, Completed the present invention by finding that it is possible to form enzyme cycling by adding and acting a benzylamine transaminase derived from a microorganism together with a substrate to amplify and generate hydrogen peroxide, ammonia, and α-keto acid to be measured. It led to.

[0008] That is, the present invention is characterized in that a benzylamine transaminase, an amino group donor, and a benzylamine oxidase are allowed to act on a sample to perform an amplification reaction by an enzyme cycling method, and the resulting amplification reaction product is quantified. Is a highly sensitive method for quantitative determination of benzylamines.

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

[0010]

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The reaction principle of enzyme cycling will be described according to the above equation. Benzylamines in a sample are first converted to benzaldehydes by a benzylamine oxidase, and hydrogen peroxide and ammonia are simultaneously produced. Next, the converted benzaldehydes are converted to the original benzylamines by the catalysis of benzylamine transaminase in the presence of an amino group donor, and at the same time, α-keto acids are formed. By repeating this series of reactions,
Hydrogen peroxide, ammonia, and α-keto acid are amplified and generated and accumulated.

It is a feature of the present invention that the amount of benzylamines in a sample is determined by quantifying at least one of these amplification reaction products.

The subject referred to in the present invention refers to a liquid such as urine, blood, serum, plasma, culture, culture solution, or intracellular solution, or an extract thereof, containing benzylamines to be quantified.

The benzylamines to be subjected to the quantification method of the present invention refer to benzylamine or those having a substituent on the phenyl ring of benzylamine.

The benzylamine having a substituent includes:
An alkyl group such as a methyl group and an ethyl group; a halogen atom such as a chlorine atom, a bromine atom and a fluorine atom; an alkoxy group such as a methoxy group; a cyano group; a nitro group; a hydroxy group; , Meta-, or para-substituted o-, m-, or p-
And substituted benzylamines. Specific examples include p-methylbenzylamine, p-methoxybenzylamine, p-chlorobenzylamine, p-fluorobenzylamine, p-carboxybenzylamine and the like.

The amino group donor in the present invention is determined by the substrate specificity of the benzylamine transaminase to be used, and is converted into α-keto acid by the action of the enzyme, and at the same time, the other substrate, benzaldehydes Is converted to benzylamines. Specific examples of the amino group donor include L-alanine, L-glutamic acid, and glycine.

The benzylamine transaminase used in the present invention comprises 1 mol of benzylamines and 1 mol of α
-Acts on keto acids to produce one mole of benzaldehyde and one mole of amino group donor, and in the reverse reaction, one mole of benzaldehyde and one mole of amino group donor Is not particularly limited as long as it produces 1 mol of benzylamines and 1 mol of α-keto acid, and an enzyme having such properties can be used without limitation.

In the present invention, enzyme cycling is formed by utilizing the above-mentioned reverse reaction of benzylamine transaminase and used for quantitative determination. .

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

[0020]

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Examples of such enzymes include the genus Enterobacter, the genus Bacillus,
Agrobacterium, Corynebacterium, Comamon
as) genus, Pseudomonas genus, Brevibacterium genus, Streptomyces genus, Actinomyces
Genus, Spirillospora, Penicillium, or Neocosmospora
and benzylamine transaminase derived from microorganisms derived from the genus cosmospora).

The benzylamine transaminase used in the present invention has a strong action on benzylamines, and has a property of hardly acting on amines and polyamines such as β-alanine and putrescine which are considered to exist in living organisms. Things are desirable. Benzylamine transaminase having properties advantageous for the determination of benzylamines in terms of substrate specificity is disclosed, for example, in Japanese Patent Application No. Hei.
Bacillus brevis B-17 described in No. 327827
5 (Bacillus brevis B-175, 13312
No. benzylamine transaminase.

The benzylamine oxidase used in the present invention is an enzyme that catalyzes the reaction of oxidizing 1 mol of benzylamines to produce 1 mol of benzaldehyde, 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 above-mentioned benzylamine oxidase may be of any origin, for example, Aspergillus (Aspergillus).
Lus), those derived from microorganisms such as those derived from the genus Penicillium, the genus Monascus, the genus Fusarium, and the genus Mucor can be used.

The benzylamine oxidase used in the present invention strongly acts on benzylamines,
Enzymes, such as alanine and putrescine, which are unlikely to act on amines and polyamines which are considered to exist in living organisms are desirable. Such benzylamine oxidases having properties advantageous for the determination of benzylamines in terms of substrate specificity include, for example, Agricultural Biological Chemistry (Agric. Biol. Chem.), Vol. 29, p. 117. (1965)
Aspergillus niger AT described in
Amine oxidase derived from CC 28325).

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

The α-keto acid amplified and generated by the enzyme cycling reaction depends on the amino acid donor determined by the substrate specificity of benzylamine transaminase used in the reaction, and the amino group of the amino group donor used is substituted with a carbonyl group. Is a compound having the structure shown below. For example, when the amino group donor is L-alanine, pyruvate is generated as 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-keto acid. Ketoglutaric acid is produced respectively.

The conditions under which benzylamine transaminase and benzylamine oxidase are allowed to act on the subject may be appropriately selected from the conventionally known conditions of the action of the enzyme, and generally, the benzylamine transaminase used is generally used. And the optimum pH and temperature of benzylamine oxidase, and conditions under which the activity of the enzyme is maximized are desirable.

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

The pH condition is usually 4.0 to 10.5.
The pH is preferably in the range of 6.5 to 8.0, and a buffer is usually used to maintain the pH in these ranges. The type of the buffer is not particularly limited, and a known buffer such as a phosphate buffer, a citrate buffer, and a Tris-HCl buffer is used. The concentration of the buffer is not particularly limited, but is preferably, for example, 2 to 500 mM.

The temperature condition is generally in the range of 15 to 60 ° C, but preferably in the range of 30 to 40 ° C.

The amounts of benzylamine transaminase and benzylamine oxidase used for quantification in the present invention vary depending on the activity of the enzyme, reaction conditions, the concentration of benzylamines to be measured, and the like, and cannot be limited unconditionally. Preferably, at a set benzylamine concentration, the amplification reaction product is amplified within a predetermined time, detected,
A sufficient amount to be quantified is good. Such an amount of benzylamine transaminase and an amount of benzylamine oxidase are usually used in the range of 0.1 to 100 U / ml, respectively, and preferably in the range of 1 to 50 U / ml.

The amount of the amino group donor used in the enzyme cycling reaction is preferably 5 to 20 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 four components of an amino group donor, benzylamines, benzylamine transaminase and benzylamine oxidase are present simultaneously. Therefore, usually, a solution containing two or three of these four components is prepared, and after reaching the reaction set conditions, the remaining one is obtained.
Alternatively, a means for initiating a cycling reaction by adding two components and subjecting it to quantitative determination is employed. Although the last component to be added is not limited, it is common to add the enzyme, benzylamine transaminase or benzylamine oxidase, or both enzymes last.

The reaction time of the enzyme cycling reaction when quantifying benzylamines varies depending on the reaction conditions, the concentration of the benzylamines to be measured, and the like, and cannot be unconditionally limited. It is desirable that the time is sufficient for the product of the enzyme cycling reaction to be amplified and the reaction to be confirmed. As the reaction time, a range of 2 minutes to 5 hours is usually adopted, but a range of 2 minutes to 30 minutes is preferable.

The termination of the enzyme cycling reaction is carried out after a predetermined time by adding a reaction stopping reagent. As a reaction terminating reagent, for example, sodium chloride or the like which is an inhibitor of benzylamine oxidase is used.

In the method for highly sensitive determination of benzylamines according to the present invention, at least one of hydrogen peroxide, ammonia and α-keto acid, which are amplification reaction products by an enzyme cycling reaction, may be detected and quantified. .

[0038] Any of the amplification reaction products obtained by the enzyme cycling can be quantified by a conventionally known method.

When the ammonia and α-keto acid are α-ketoglutarate, an enzymatic method using L-glutamate dehydrogenase and NADH, and when the α-keto acid is pyruvic acid, an enzymatic method using L-lactate dehydrogenase and NADH. In the case where the α-keto acid is oxaloacetic acid, it can be determined by an enzymatic method using oxaloacetate decarboxylase.

The determination of hydrogen peroxide can be easily carried out, for example, by the conventionally known 4-aminoantipyrine-peroxidase method. By using a chemiluminescence method for the determination of hydrogen peroxide, it is possible to use a more sensitive measurement method.

When using any of the quantification methods, a dilution series of a benzylamine solution having a known concentration is prepared, and benzylamine transaminase and benzylamine oxidase are allowed to act on the solution of the dilution series. Any one or more of certain hydrogen peroxide, ammonia and α-keto acid are quantified by the above-mentioned method, and a calibration curve between the amount of benzylamines and the amount of amplification reaction product is prepared.
Next, the amount of the amplification product is obtained for the specimen by the same method, and the amount of benzylamines in the specimen can be obtained from the calibration curve obtained previously.

When the amount of benzylamines in the sample is about 1 mM or less, the amount of benzylamine as a substrate is proportional to the production rate of the amplification reaction product by the enzyme cycling reaction. The amount of benzylamines in the sample can be determined from the rate of amplification reaction product formation. That is, a dilution series of a benzylamine solution having a known concentration is prepared, and benzylamine transaminase and benzylamine oxidase are allowed to act on the solution of the dilution series, whereby hydrogen peroxide, ammonia and α-keto which are amplification reaction products are produced. The production rate of one or more of the acids is measured by the above-mentioned method, and a calibration curve is prepared between the amount of benzylamines and the production rate of the amplification reaction product. Next, an amplification product generation rate is obtained in the same manner for the analyte,
The amount of benzylamines in the sample can be determined from the calibration curve obtained previously.

[0043]

According to the method for quantifying benzylamines according to the present invention, benzylamine serving as a substrate is subjected to enzyme cycling by utilizing the action of benzylamine transaminase and benzylamine oxidase in the presence of an amino group donor. It is characterized by quantifying an amplification reaction product amplified and generated as a result of the enzyme cycling reaction.

[0044]

The method for highly sensitive determination of benzylamines according to the present invention can be carried out without considering the low sensitivity of the conventional paper chromatography method, performing complicated operations, and performing complicated maintenance of the high performance liquid chromatography method. The determination of benzylamines can be performed. In addition, as compared with the conventional measurement method using benzylamine oxidase, it is possible to obtain about ten times the sensitivity by performing enzyme cycling. Therefore, it can be quantified even when the amount of benzylamines in the subject is very small. As a result, quantification of benzylamines can be carried out easily, in a short time, and with high accuracy as a daily work.

[0045]

EXAMPLES 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,
Sterilize by dispensing 1,500 ml of a medium consisting of 0.1% salt, 0.1% benzylamine, 0.02% antifoam (Ainol), pH 7.0 (120
A Bacillus brevis B-175 microtechnical laboratory No. 13312) was inoculated into a 5,000 ml Erlenmeyer flask kept at 20 ° C. for 20 minutes). After performing shaking culture at 28 ° C. for 72 hours,
The main culture was carried out by adding 20 l of a medium having the same composition as above to a jar fermenter previously steam-sterilized. After culturing for 70 hours at 28 ° C., a stirring frequency of 150 rpm, and aeration speed of 20 l / min, the culture solution was centrifuged to collect cells. About 150 g (wet cell weight) of the obtained cells was suspended in 500 ml of 10 mM phosphate buffer (pH 7.5), and the suspension was disrupted by an ultrasonic disrupter. The crushed liquid was centrifuged using a centrifuge to obtain a supernatant. The total activity of benzylamine transaminase in this supernatant was 5,100 units, and the specific activity was 0.14 units / mg-protein.

A column (φ) packed with DEAE-cellulose (trade name: DE-52 manufactured by Whatman), which was obtained by preliminarily equilibrating the supernatant with a 20 mM phosphate buffer (pH 7.5).
7x40 cm) to adsorb the enzyme. This column is 10 mM
5,000 phosphate buffer (pH 7.5) containing ammonium sulfate
After washing with ml, a linear concentration gradient of a similar phosphate buffer with an ammonium sulfate concentration of 10 mM to 230 mM (total volume: 10 l)
The protein adsorbed was eluted in the above step, and a benzylamine transaminase active fraction was recovered. The total activity of benzylamine transaminase in this active fraction was 1,400 units, and the specific activity was 1.25 units / mg-protein.

The active fraction was desalted and concentrated using an ultrafiltration apparatus, and then Sephacryl S-400 (Pharmacia) previously equilibrated with 20 mM phosphate buffer (pH 7.5) containing 0.2 M ammonium sulfate. The product was passed through a column (φ6.5 × 120 cm) packed with the product and subjected to gel filtration to collect an active fraction. The total activity of benzylamine transaminase in this active fraction was 970 units, and the specific activity was 2.1 units / mg-tank.

The above purified enzyme was prepared by adding 10 mM phosphate buffer (pH 7.5)
Substrate specificity, optimal pH, pH stability, optimal temperature, and temperature stability of the present enzyme were examined using an enzyme preparation prepared by appropriately diluting the above.

(Substrate specificity) 0.2 M phosphate buffer (pH 7.5)
0.74 ml, 0.1 mM 50 mM pyruvate solution, 0.06 ml 5 mM pyridoxal phosphoric acid solution, and 0.04 ml of 100 mM solution of amines or amino acids serving as various amino group donors.
To a reaction solution consisting of 5 ml, 0.05 ml (0.05 unit) of an enzyme standard was added and reacted at 30 ° C. for 30 minutes. After the reaction solution was immersed in a boiling water bath for 30 seconds to inactivate the enzyme, the precipitate was removed by centrifugation to obtain a clear transparent solution. This clear solution was injected into liquid chromatography to quantitatively analyze L-alanine. Table 1 shows the strength of the action of benzylamine transaminase on these amino group donors, expressed as a total activity value with the action on benzylamine being 100%. When pyruvate was used as an amino group acceptor, this benzylamine transaminase showed the strongest action on benzylamine as an amino group donor and was found to be an enzyme that did not act on β-alanine and putrescine.

[0051]

[Table 1]

(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:
0.5 ml of Tris-HCl buffer; pH 9.0 to 11.5: glycine-sodium hydroxide buffer), 0.1 ml of a 5 mM pyridoxal phosphate solution, 0.1 ml of a 50 mM pyruvic acid solution, and 0.05 ml of a 100 mM benzylamine solution. Enzyme preparation in a reaction solution consisting of ml
0.1 ml (0.05 unit) was added and reacted at 37 ° C. for 5 minutes. After the reaction solution was immersed in a boiling water bath for 30 seconds to inactivate the enzyme, the precipitate was removed by centrifugation to obtain a clear transparent solution. This clear solution was injected into liquid chromatography to quantitatively analyze L-alanine to determine the enzyme activity.
FIG. 1 was obtained by calculating the relative activity value with the maximum enzyme activity value taken as 100%. FIG. 1 shows that the optimum pH of the present enzyme is in the range of 7.0 to 9.0.

(PH stability) 0.2 M various buffers (pH 4.5 to 6.
5: citrate buffer; pH 6.5-8.0: phosphate buffer; pH 7.5-9.
0: Tris-HCl buffer; pH 8.0-0.5: Glycine-sodium hydroxide buffer) 0.95 ml / 0.05 ml of enzyme preparation (0.4 unit)
And left at 30 ° C for 60 minutes.
0.5 ml of 0.2 M phosphate buffer (pH 8.0), 50 mM pyruvate solution
The mixture was dispensed and mixed with an activity measurement solution consisting of 0.1 ml, 0.1 mM of a 5 mM pyridoxal phosphate solution, and 0.05 ml of a 100 mM benzylamine solution, and reacted at 37 ° C. for 5 minutes. After the reaction solution was immersed in a boiling water bath for 30 seconds to inactivate the enzyme, the precipitate was removed by centrifugation to obtain a clear transparent solution. This clear solution was injected into liquid chromatography to quantitatively analyze L-alanine to determine the enzyme activity. Maximum enzyme activity value of 10
FIG. 2 was obtained by calculating the relative activity value with 0%. As is clear from FIG. 2, the present enzyme is stable in the pH range of 5.5 to 8.5.

(Optimal temperature) 0.2 M phosphate buffer (pH 8.0) 0.5
0.1 ml (0.05 unit) of the enzyme sample was added to an activity measurement solution consisting of 0.1 ml of a 50 mM pyruvate solution, 0.1 ml of a 5 mM pyridoxal phosphate solution, and 0.05 ml of a 100 mM benzylamine solution. , 37, 40, 45, 50, 55, 60, 65, 70, 75 and 80 ° C. at each temperature for 5 minutes. After the reaction solution was immersed in a boiling water bath for 30 seconds to inactivate the enzyme, the precipitate was removed by centrifugation to obtain a clear transparent solution. This clear solution was injected into liquid chromatography to quantitatively analyze L-alanine to determine the enzyme activity. Maximum enzyme activity
Fig. 3 was obtained by calculating the relative activity value as 100%. FIG. 3 shows that the optimum temperature of the present enzyme is in the range of 45 to 50 ° C.

(Temperature stability) 10 mM phosphate buffer (pH 7.5)
0.2 ml (0.04 units) of the enzyme standard diluted in 25, 30, 35, 40, 45,
The treatment was performed at 50, 55, 60, 65 and 70 ° C. for 10 minutes. 0.05 ml of this enzyme solution is 0.5 ml of 0.2 M phosphate buffer (pH 8.0), 0.1 ml of 50 mM pyruvate solution, and 5 mM of pyridoxal phosphate solution.
The mixture was dispensed and mixed with an activity measurement solution consisting of 1 ml and 0.05 ml of a 100 mM benzylamine solution, and reacted at 37 ° C. for 5 minutes. After immersing the reaction solution in a boiling water bath for 30 seconds to inactivate the enzyme, the precipitate was removed by centrifugation to obtain a clear transparent solution. This clear solution was injected into liquid chromatography to quantitatively analyze L-alanine to determine the enzyme activity. FIG. 4 was obtained by calculating the relative activity value with the maximum enzyme activity value taken as 100%. As is evident from FIG. 4, the 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 a 10 mM phosphate buffer (pH 7.5) and an agricultural biological chemistry (Agric. Biol. Chem.), 29, 117
Aspergillus niger (1965)
niger ATCC28325), using a benzylamine oxidase prepared by a known method, a calibration curve showing the correlation between the benzylamine concentration and the amount of hydrogen peroxide generated as one of the amplification reaction products was obtained by the following method. Created.

In a 1 cm optical path width cuvette, 200 mM L-alanine, 0.3 mM pyridoxal phosphate, 1 mM N-ethyl-N
20 mM phosphate buffer containing sodium-(-2-hydroxy-3-sulfopropyl) -m-toluidine, 1 mM 4-aminoantipyrine, 10 U / ml peroxidase, 0,2,10,20,50 μM of each benzylamine 0.95 ml of the solution (pH 7.5) was added, and incubation was performed at 30 ° C. for 10 minutes. In each of these cuvettes, 2 U / ml of benzylamine transaminase and 0.5 U / ml of benzylamine oxidase obtained in Production Example 1 were added.
0.05 ml was mixed and reacted at 30 ° C. for 20 minutes, the absorbance at 555 nm corresponding to the increase in hydrogen peroxide was measured, and a calibration curve showing the relationship between the benzylamine concentration and the absorbance was obtained (FIG. 5). . As a comparative example, when the enzyme cycling was not performed, the absorbance was measured under the same conditions without adding benzylamine transaminase (FIG. 5).

From FIG. 5, it can be seen that the calibration curve of benzylamine has good linearity, that the method of the present invention can be used for accurate quantification of benzylamine, and has a sensitivity about 10 times that when no enzyme cycling reaction is performed. Understand.

Example 2 An enzyme preparation prepared by diluting the purified benzylamine transaminase obtained in Production Example 1 with a 10 mM phosphate buffer (pH 7.5) and an agricultural biological chemistry (Agric. Biol. Chem.), 29, 117
Aspergillus niger (1965)
niger ATCC 28325), and a calibration curve showing the correlation between the p-fluorobenzylamine concentration and the amount of generated hydrogen peroxide was prepared by the following method using benzylamine oxidase prepared by a known method.

In a 1 cm optical path width cuvette, 200 mM L-alanine, 0.3 mM pyridoxal phosphate, 1 mM N-ethyl-N
20 mM with sodium-(-2-hydroxy-3-sulfopropyl) -m-toluidine, 1 mM 4-aminoantipyrine, 10 U / ml peroxidase, 0,2,10,20,50 μM of each p-fluorobenzylamine 0.95 ml of a phosphate buffer (pH 7.5) was added, and incubation was performed at 30 ° C. for 10 minutes. Each of these cuvettes was mixed with 2 U / ml of benzylamine transaminase obtained in Production Example 1 and 0.05 ml of 0.5 U / ml of benzylamine oxidase, and reacted at 30 ° C. for 20 minutes to increase the amount of hydrogen peroxide. The corresponding absorbance at 555 nm was measured, and a calibration curve showing the relationship between the benzylamine concentration and the absorbance was obtained (FIG. 6). As a comparative example, when the enzyme cycling was not performed, the absorbance was measured under the same conditions without adding benzylamine transaminase (FIG. 6).

FIG. 6 shows that the calibration curve of p-fluorobenzylamine has good linearity, and the method of the present invention can be used for accurate quantification of p-fluorobenzylamine, which is about 10 times that when the enzyme cycling reaction is not performed. It can be seen that the sensitivity is as follows.

[Brief description of the drawings]

FIG. 1 is a diagram showing the optimum pH of benzylamine transaminase used in Examples.

FIG. 2 is a diagram showing the pH stability of benzylamine transaminase used in Examples.

FIG. 3 is a diagram showing the optimum temperature of benzylamine transaminase used in Examples.

FIG. 4 is a diagram showing the temperature stability of benzylamine transaminase used in Examples.

FIG. 5 shows a benzylamine quantification calibration curve prepared according to the present invention and a benzylamine quantification calibration curve prepared as a comparative example.

FIG. 6 shows a calibration curve for p-fluorobenzylamine quantification prepared according to the present invention and a calibration curve for p-fluorobenzylamine quantification prepared as a comparative example.

Claims (1)

(57) [Claims] Claims: 1. An amplification reaction by an enzyme cycling method is carried out by allowing a benzylamine transaminase, an amino group donor, and a benzylamine oxidase to act on a subject,
A highly sensitive method for quantifying benzylamines, which comprises quantifying the resulting amplification reaction product.