JPH07184693A - Method for measuring activity of benzylamine transaminase and reagent for measuring enzymic activity - Google Patents

Abstract

PURPOSE:To simply measure the subject enzyme which is a metabolic enzyme of catecholamines with a high accuracy by reacting a test specimen with an alpha-keto acid and benzylamine, then reacting the resultant product with an oxidized type NAD<+> and a dehydrogenase and determining the formed reduced type NADH. CONSTITUTION:This method for simply measuring the objective activity of an enzyme which is a metabolic enzyme of catecholamines in the living body with a good accuracy in a short time is to react a test specimen such as urine, blood, blood serum, a culture, an intracellular liquid, etc., containing a benzylamine transaminase which is an object of measurement with an alpha-keto acid (e.g. pyruvic acid) and benzylamine, react the formed benzaldehyde with a benzaldehyde dehydrogenase in the presence of an oxidized type nicotinamide coenzyme [e.g. nicotinamide adenine dinucleotide (NAD<+>)], measure the absorbance at 340nm and determine the reduced type nicotinamide coenzyme (e.g. NADH) produced by the reaction in measuring the activity of the benzylamine transaminase in the test specimen.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for measuring the activity of benzylamine transaminase in a subject. INDUSTRIAL APPLICABILITY The present invention provides a method for enzymatically and simply and accurately measuring the enzymatic activity of benzylamine transaminase in a sample, and an enzyme activity measuring reagent therefor.

[0002]

PRIOR ART Benzylamine transaminase is
It acts as a metabolic enzyme of catecholamines that is carried out in vivo, and by measuring the enzymatic activity of this benzylamine transaminase, it is possible to obtain important knowledge in the study of the metabolism and physiological actions of catecholamines.

As a method for measuring the activity of benzylamine transaminase, conventionally, a method using an amino acid analyzer has been generally used. According to this method, the analyte is allowed to react with benzylamine in the presence of pyruvic acid for a certain period of time, and alanine produced as the enzymatic reaction proceeds is quantitatively analyzed to obtain the benzylamine transaminase activity value in the analyte. .

For example, Agricultural Biological Chemistry (Agric. Biol. Che)
m. ), 42, 2363 (1978), the activity of benzylamine transaminase is measured by the following complicated method. That is,
After adding the analyte containing benzylamine transaminase to benzylamine in the presence of pyruvic acid and allowing the reaction to proceed for a certain period of time, the enzymatic reaction is stopped by adding a protein denaturant or heat treatment, and further centrifuging the reaction solution. Centrifuge using a machine to collect the supernatant.
L-alanine, which is one of the products produced by the enzymatic reaction of benzylamine transaminase in the supernatant, is quantified by an amino acid analyzer to measure the activity value of benzylamine transaminase in the test sample.

[0005]

[Problems to be Solved by the Invention] A method using an amino acid analyzer which has been generally used as a method for measuring benzylamine transaminase activity requires complicated enzyme reaction itself and an expensive analyzer body. In addition, there are many problems and problems such as maintenance / replacement of the packed column and corrosion around the liquid delivery pump due to high ionic strength of the developing solution. Furthermore, it is difficult to make the analysis accuracy of enzyme activity measurement 2% or less in terms of simultaneous reproducibility and day difference reproducibility.

[0006]

Means for Solving the Problems The inventors of the present invention have earnestly studied a simple and highly accurate method for measuring the activity of benzylamine transaminase in order to solve the problems in the conventional method for measuring the activity. As a result, we focused on benzaldecht, which is one of the reaction products generated by reacting a test substance with α-keto acid and benzylamine, and this benzaldecht is a benzaldehyde dehydrogenase in the presence of oxidized nicotinamide coenzyme. It was found that the oxidized nicotinamide coenzyme is converted into a reduced nicotinamide coenzyme which is easily detected by the action of benzylamine. Has completed the method for measuring the activity of.

That is, in the present invention, when measuring the activity of benzylamine transaminase in a test sample, α-keto acid and benzylamine are allowed to act on the test sample, and the benzaldehyde produced by the presence of oxidized nicotinamide coenzyme. A method for measuring the activity of benzylamine transaminase, which comprises reacting benzaldehyde dehydrogenase and quantifying the reduced nicotinamide coenzyme produced in the reaction.

The term "analyte" as used in the present invention refers to a liquid or extract containing benzylamine transaminase to be measured, such as urine, blood, serum, plasma, culture, culture fluid, or intracellular fluid.

The benzylamine transaminase which is the object of the activity measuring method of the present invention is benzylamine and α
-An enzyme that catalyzes the reaction that produces benzaldehyde and α-amino acids from keto acids.

The α-keto acid used in the present invention is not particularly limited, and it is sufficient to select one which is recognized by the benzylamine transaminase used as a substrate. Typical α-keto acids include pyruvic acid, α-ketoglutaric acid, glyoxalic acid and the like.

The oxidized nicotinamide coenzyme used in the present invention is not particularly limited, and any one can be used as long as it can be recognized by the benzaldehyde dehydrogenase used as a substrate. For example, nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD ⁺ ), nicotinamide adenine dinucleotide phosphate (hereinafter N
ADP ⁺ ), thionicotinamide adenine dinucleotide (hereinafter abbreviated as Thio-NAD ⁺ ), and thionicotinamide adenine dinucleotide phosphate (abbreviated as Thio-NADP ⁺ ).

The benzaldehyde dehydrogenase used in the present invention acts on 1 mol of benzaldehyde and 1 mol of oxidized nicotinamide coenzyme, and acts on 1 mol of benzyl alcohol and 1 mol of reduced nicotinamide coenzyme. There is no particular limitation as long as it produces a substance, and an enzyme having such properties can be used without particular limitation. The action of this benzaldehyde dehydrogenase is shown below.

Oxidized nicotinamide coenzyme + benzaldehyde-> reduced nicotinamide coenzyme + benzyl alcohol As an example of such an enzyme, Journal of Bacteriology, 66.
Volume 548-553 (1953). Pseudomonas f.
benzaldehyde dehydrogenase, Journal of Biological Chemistry (J. Biol. Chem.), 242, 5294.
Pseudomonas described on page 5300 (1967)
Fluorescence A3.12 (Psudomonas
Fluorescens A3.12, ATCC 1
2633) -derived benzaldehyde dehydrogenase, and Pseudomonas fluorescens (Pseudomo
NAS Fluorescens IFO 3903)
Examples thereof include benzaldehyde dehydrogenase derived from the above.

Known conditions are employed without particular limitation as conditions for allowing the test substance to act on α-keto acid, benzylamine, oxidized nicotinamide coenzyme, and benzaldehyde dehydrogenase. In general, it is desirable to carry out the treatment at around the optimum pH and the optimum temperature of benzylamine transaminase in the analyte to be measured so that the enzyme activity is maximized.

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

The pH condition is preferably 4.0 to 10.5, and it is general to use a buffer solution capable of maintaining the pH in this range. The type of the buffer solution is not particularly limited, but examples of suitable buffer solutions include a phosphate buffer solution, a citrate buffer solution, a Tris-hydrochloric acid buffer solution, and a Good's buffer solution. The concentration of these buffers is not particularly limited, but a concentration range of, for example, 2 to 500 mM is suitable.

The temperature condition of the activity measuring method of the present invention is preferably in the range of 15 to 40 ° C.

The concentration of α-keto acid varies depending on the type of benzylamine transaminase enzyme present in the subject, but the concentration is 2 to 100 times the Km value,
That is, it is used in a concentration range of 0.5 to 400 mM, but particularly in a concentration range of 4 to 25 times, that is, 1 to 100 m.
A concentration range of M is suitable.

The concentration of benzylamine used also varies depending on the type of benzylamine transaminase enzyme present in the subject, but is in the concentration range of 2 to 50 times the Km value of the enzyme relative to benzylamine, that is, 0.5 to.
Although it is used in a concentration range of 200 mM, a concentration range of 4 to 20 times, that is, a concentration range of 1 to 80 mM is particularly preferable.

The concentration of the oxidized nicotinamide coenzyme used in the present measuring method depends on the properties of the benzaldehyde dehydrogenase used, but in general, the Km value of the benzaldehyde dehydrogenase for the oxidized nicotinamide coenzyme is 2 ~ 200 times concentration range, i.e. 0.5 ~ 200m
It is added in a concentration range of M, but a concentration range of 4 to 100 times, that is, a concentration range of 1 to 100 mM is particularly preferable.

Further, the amount of benzaldehyde dehydrogenase used in the measuring method of the present invention will be described. The amount of the enzyme is shown below as 1 U or 1 unit of the enzyme that produces 1 μmole of product per minute, and the enzyme concentration is shown as U / ml as the number of units per 1 ml of the reaction solution. The amount of benzaldehyde dehydrogenase used in the measurement method of the present invention varies depending on the activity of the enzyme, the reaction conditions, the concentration of benzylamine transaminase to be measured, and the like, and cannot be unconditionally limited, but preferably benzyl present in the test sample. Benzaldehyde produced by the enzymatic reaction of amine transaminase is completely converted into benzyl alcohol within a predetermined time, and sufficient amount of benzaldehyde dehydrogenation to convert its chemically equivalent amount of oxidized nicotinamide coenzyme to reduced nicotinamide coenzyme. Good amount of enzyme. The amount of such benzaldehyde dehydrogenase is used in the range of 0.01 to 100 U / ml, and particularly preferably in the range of 0.05 to 50 U / ml.

The reaction in the measuring method of the present invention can be easily detected in the reduced nicotinamide only when four components of α-keto acid, benzaldehyde, oxidized nicotinamide coenzyme and benzaldehyde dehydrogenase are present at the same time. Production of coenzyme begins. Therefore, after reacting the analyte with a mixed solution of α-keto acid and benzylamine in advance to generate benzaldehyde, the solution containing two components of oxidized nicotinamide coenzyme and benzaldehyde dehydrogenase is added to the analyte. It is also possible to measure the benzylamine transaminase activity in a sample. However, in order to improve the accuracy of the measured value of the benzylamine transaminase enzyme activity in the subject, α-keto acid, benzylamine, oxidized nicotinamide coenzyme,
It is preferable to simultaneously mix the benzaldehyde dehydrogenase and the analyte to start the reaction. Such a measuring method will be described in detail later, but a solution consisting of four components of α-keto acid, benzylamine, oxidized nicotinamide coenzyme, and benzaldehyde dehydrogenase is prepared as a reagent in advance, and the reaction is performed. A method in which the reaction is started by adding a test substance after reaching the set conditions is suitable.

The reaction time in the measuring method of the present invention varies depending on the reaction conditions, the concentration of benzylamine transaminase in the sample to be measured, and the like, and it cannot be limited to any specific one, but preferably it is converted under the set conditions. It is desirable that the time is sufficient for confirming the reduced nicotinamide coenzyme. The reaction time is preferably 2 minutes to 5 hours.

As a method for quantifying reduced nicotinamide coenzyme, an existing general method can be used. For example, when the reduced nicotinamide coenzyme produced is NADH or NADPH, the reduced nicotinamide coenzyme produced produces an increase in absorbance at 340 nm.
-NADH or Thio-NADPH can be quantified by measuring the increase in absorbance at 400 nm. For highly sensitive measurement, the reduced nicotine amide coenzyme and the tetrazolium salt compound are generated to form a formazan dye by the action of diaphorase, and the formazan dye is colorimetrically determined with high sensitivity. The amount of amide coenzyme produced can be quantified.

The reagent for measuring the activity of benzylamine transaminase of the present invention is a reagent composed of four components of α-keto acid, benzylamine, oxidized nicotinamide coenzyme and benzaldehyde dehydrogenase. The concentration range of each component contained in the reagent will be described below in the case where 20 parts by volume of the activity measuring reagent is mixed with 1 part by volume of the analyte.

The α-keto acid is contained in a concentration range of 0.5 to 400 mM, but a concentration range of 1 to 100 mM is particularly preferable. Benzylamine is contained in a concentration range of 0.5 to 200 mM, but a concentration range of 1 to 80 mM is particularly preferable. Oxidized nicotinamide coenzyme is 0.5 to 2
It is added in a concentration range of 00 mM, but especially 1 to 100 m
A concentration range of M is suitable. Further, the benzaldehyde dehydrogenase is contained in the range of 0.01 to 100 U / ml, but the range of 0.05 to 50 U / ml is particularly preferable.

The activity measuring reagent is preferably in the form of an aqueous solution dissolved in a buffer solution in order to maintain the pH range as shown in the above-mentioned measuring method. The type or concentration of the buffer solution varies depending on the type and concentration of the benzylamine transaminase to be measured or the analyte used for the measurement, but generally the pH can be maintained at 4.0 to 10.5 during the reaction. A phosphate buffer solution, a citrate buffer solution, a Tris-hydrochloric acid buffer solution, a Good's buffer solution and the like can be appropriately used. The concentration of these buffers is not particularly limited, but a concentration range of, for example, 2 to 500 mM is suitable.

[0028]

The method for measuring the activity of benzylamine transaminase according to the present invention comprises adding the analyte in the presence of benzylamine, which is a substrate of benzylamine transaminase in the analyte, and α-keto acid, and converting the benzaldehyde produced into oxidized nicotine. A method for measuring the activity of benzylamine transaminase and a reagent for measuring the activity, which comprises allowing benzaldehyde dehydrogenase to act in the presence of an amide coenzyme to quantify the reduced nicotinamide coenzyme produced.

[0029]

INDUSTRIAL APPLICABILITY The method for measuring the activity of benzylamine transaminase according to the present invention is different from the method using an amino acid analyzer which requires a complicated maintenance of the equipment, which has been conventionally performed, and is easy to use. It has become possible to measure the activity of. Therefore, the activity of benzylamine transaminase can be easily measured in a short time with high accuracy as a daily work.

[0030]

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 0.2% glucose, 1.0% polypeptone, 0.2%
Sterilize by dispensing 1,500 ml of a medium consisting of yeast extract, 0.1% sodium chloride, 0.2% mandelic acid, 0.02% antifoaming agent (Ainol) and pH 7.0.
Pseudomonas f (Pseudomonas f) in a 5,000 ml Erlenmeyer flask
lourescens IFO 3903). 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 conditions were 28 ° C., the number of stirrings was 150 rpm, and the aeration rate was 20 liter / min. After 70 hours of culture, the culture solution was centrifuged to collect bacterial cells. About 120 g of the obtained bacterial cells (wet bacterial cell weight) was added to 10 mM.
The suspension was suspended in 500 ml of a phosphate buffer (pH 7.5), and the suspension was disrupted by an ultrasonic disrupter. The disrupted liquid was centrifuged using a centrifuge to obtain a supernatant liquid. The total activity of benzaldehyde dehydrogenase in this supernatant was 2,300 units, and the specific activity was 0.025 units / mg-protein.

This 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. This column is loaded with 20 mM phosphate buffer (pH 7.5) 5,0
After washing with 00 ml, ammonium sulfate concentration is 0 to 50
The adsorbed protein was eluted with a similar linear concentration gradient (total volume: 10 liters) of a phosphate buffer of 0 mM, and a benzaldehyde dehydrogenase active fraction was collected.
The total activity of benzaldehyde dehydrogenase in this active fraction was 800 units, and the specific activity was 0.52 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 benzaldehyde dehydrogenase in this active fraction was 360 units, and the specific activity was 2.1 units / mg-protein.

Example 1 First, an enzyme activity measuring reagent was prepared using the benzaldehyde dehydrogenase-producing enzyme obtained in Production Example 1, and then Bacillus brevis (B-175) was used as a test sample.
Microcentrifuge Microbial Co. No. 13312) was sonicated to prepare a centrifugal supernatant solution, and the activity of benzylamine transaminase in the solution was measured.

[Preparation of Reagent for Measuring Enzyme Activity] 20 mM Pyruvate 5 mM Benzylamine Sulfate 5 mM NAD ⁺ 5 U / ml Benzaldehyde Dehydrogenase 50 mM Phosphate Buffer (pH 7.5) And

[Measurement of Benzylamine Transaminase Activity] 2.0 ml of the above enzyme activity measurement reagent was poured into a cuvette having an optical path width of 1 cm, and heated at 30 ° C. for 2 minutes. Then, 100 μl of the test sample was dispensed and mixed, and immediately after incubating at 30 ° C., 340
The absorbance per nm was followed to quantify the amount of NADH produced per minute. The benzylamine transaminase activity in the test sample was calculated from the production rate of NADH. The same operation was performed 5 times in total, and the coefficient of variation (CV value), which is an index of simultaneous reproducibility, was calculated to test the accuracy of activity measurement. The results are shown in Table 1.

[0037]

[Table 1]

Example 2 The same enzyme activity as in Example 1 was measured in the same manner as in Example 1 except that NAD ⁺ used in the example was replaced with Thio-NAD ⁺ .

[Preparation of Reagent for Measuring Enzyme Activity] 20 mM Pyruvate 5 mM Benzylamine Sulfate 5 mM Thio-NAD ⁺ 5 U / ml Benzaldehyde Dehydrogenase 50 mM Phosphate Buffer (pH 7.5) Use as measurement reagent.

[Measurement of Benzylamine Transaminase Activity] 2.0 ml of the above enzyme activity measuring reagent was poured into a cuvette having an optical path width of 1 cm, and heated at 30 ° C. for 2 minutes. Then, 100 μl of the same test substance as in Example 1 was dispensed and mixed, and immediately after the incubation at 30 ° C., the absorbance at 400 nm was traced to generate Thio-
The amount of NADH per minute was quantified. This Thio-
The benzylamine transaminase activity in the test substance was calculated from the production rate of NADH. The same operation was performed 5 times in total, and the coefficient of variation (CV value), which is an index of simultaneous reproducibility, was calculated to test the accuracy of activity measurement. The results are shown in Table 2.

[0041]

[Table 2]

Comparative Example 1 As a comparative example, the results of the enzyme activity measuring method using an amino acid analyzer which has been conventionally used are shown. A solution consisting of 50 mM phosphate buffer (pH 7.5), 20 mM pyruvic acid, and 5 mM benzylamine sulfate was added to 1.0 m.
It was dispensed into a test tube and heated at 30 ° C. for 5 minutes. The same test object used in Examples 1 and 2 was added to this test tube.
After injecting μl and mixing, the mixture was incubated at 30 ° C. for 20 minutes. Place the reaction test tube in a boiling water bath for 30
The activity of benzylamine transaminase was inactivated by soaking for 2 seconds. After removing the precipitate with a centrifuge,
The clear solution was injected into an amino acid analyzer for L-
Alanine was quantified. The enzyme activity value of benzylamine transaminase in the test sample was calculated from the amount of L-alanine produced. This operation was repeated 5 times in total to test the accuracy of the activity measurement method. The results are shown in Table 3.

[0043]

[Table 3]

Claims (2)

[Claims] 1. When measuring the activity of benzylamine transaminase in a test sample, α-keto acid and benzylamine are allowed to act on the test sample, and benzaldehyde produced is reacted with benzaldehyde dehydrogenase in the presence of an oxidized nicotinamide coenzyme. Of benzylamine transaminase, which comprises quantifying the reduced nicotinamide coenzyme produced by the reaction 2. A reagent for measuring benzylamine transaminase activity, which comprises α-keto acid, benzylamine, oxidized nicotinamide coenzyme, and benzaldehyde dehydrogenase.