JPH0242479B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel method for quantifying agmatine using an enzyme that oxidizes agmatine (hereinafter referred to as agmatine oxidase). More specifically, the present invention provides a novel method for quantifying agmatine using a novel enzyme, agmatine oxidase. While investigating the many enzymes produced by Penicillium chrysogenum, the present inventors learned that there was a special enzyme among them, and upon investigating its properties, found that this enzyme selectively oxidizes agmatine. We confirmed that it was a new enzyme, agmatine oxidase, and established a new method for quantifying agmatine using this agmatine oxidase. Generally, agmatine is a substance produced by decarboxylation of arginine, and is an important substance on the polyamine biosynthesis pathway. Furthermore, it is known that agmatine becomes a strong mutagenic substance when nitrosated, and it is extremely important and necessary to know the amount of agmatine in living organisms or in various substances. However, the only analytical methods for agmatine that have been reported to date are chemical analysis methods that use thin layer chromatography, paper chromatography, amino acid analyzers, gas chromatography, etc., and require expensive equipment and long time. This method has the disadvantage of requiring hours of analysis time, which has been a major hindrance to the progress of agmatine analysis. In view of these circumstances, the present inventors have conducted various studies to establish a method for industrially producing an enzyme that reacts with agmatine in large quantities at low cost in order to quantify agmatine using an enzyme. , spermidine, putretsucine, agmatine, etc. as a single carbon and nitrogen source, Penicillium chrysogenum IFO4626, which can be grown as a single carbon or single nitrogen source, is cultured in a medium containing spermine, spermidine, putretsucine, agmatine, etc., We discovered that a significant amount of a novel agmatine oxidizing enzyme was produced and accumulated in culture, named it agmatine oxidase, and completed the method for quantifying agmatine of the present invention using this agmatine oxidase. be. The agmatine oxidase used in the present invention is a novel enzyme that has substrate specificity for agmatine, acts slightly on other diamines, but does not substantially act on polyamines. Agmatine oxidase used in the present invention has the following physical and chemical properties. 1 Action: As shown in the following formula, it acts on agmatine to produce 1 mol of γ-guanidinobutyraldehyde, 1 mol of ammonia, and 1 mol of hydrogen peroxide from 1 mol of agmatine. (b) Confirmation of production of hydrogen peroxide. Agmatine oxidase is allowed to act on agmatine in the presence of an enzyme, and then the enzyme system is treated with peroxidase, 4-aminoantipyrine,
When phenol is added and reacted, quinoneimine dye is generated in the reaction system (hydrogen peroxide and peroxidase, 4-aminoantipyrine,
For the reaction of phenol, see Clin.Chem.20
Volume 470 (1974)). (b) Confirmation of production of ammonia When agmatine oxidase is allowed to act on agmatine in the presence of oxygen, and then sodium hypochlorite, phenol, sodium hydroxide, and sodium nitroprusside are added to the enzyme system and allowed to react, indigo is added to the reaction solution system. Phenol is formed (for the reaction of ammonia with sodium hypochlorite, phenol, sodium hydroxide, and sodium nitroprusside, see J.
Clin. Path. vol. 13, p. 156 (1960). (c) Confirmation of production of γ-guanidinobutyraldehyde Agmatine oxidase is allowed to act on agmatine in the presence of oxygen, and potassium permanganate is then added to the enzyme system to oxidize the aldehyde produced. As a result of analyzing this solution by thin layer chromatography shown below, the oxide of the aldehyde produced was identified as γ-guanidinobutyric acid, so the product before oxidation with potassium permanganate was γ-guanidinobutyraldehyde. was identified. The thin layer plate used was silica gel 60-F-254 (manufactured by Merck & Co., West Germany), and the developing solvents were solvent system 1 [0.1 molar phosphate buffer PH7.0] and solvent system 2 [butanol:acetic acid:water = 4:1]. :5 (capacity ratio)]. After development, ninhydrin reaction and Sakaguchi reaction were performed and it was confirmed that the Rf value and color tone matched those of the standard sample. (d) Confirmation of oxygen absorption amount The consumption of oxygen in the system in which agmatine was treated with agmatine oxidase was measured using an oxygen electrode. As a result, it was confirmed that oxygen was absorbed in an amount commensurate with the amount of hydrogen peroxide produced. 2. Substrate specificity Table 1 shows the measurement results of the relative activity against other substrates when the activity against agmatine is set as 100. The activity was determined by measuring the generated hydrogen peroxide using peroxidase, phenol, and 4-aminoantipyrine methods. The substrate concentration was 2mM in both cases.

【table】

[Table] 3. Optimal PH: Around PH6.5-7.0 (as shown in Figure 1). 4 PH stability When treated at 40°C for 20 minutes, it has a residual activity of 90% or more at pH 5.5 to 7.5 (as shown in Figure 2). 5. Optimum temperature is around 45℃ at PH7.0 (as shown in Figure 3). 6 Temperature stability: Almost 100% even when treated at 40℃ for 20 minutes at PH7.0
activity remains (as shown in Figure 4). 7 Effects of inhibitors and metal ions (a) Table 2 shows the effects of various inhibitors.

【table】

[Table] * Parachloromercury benzoate ** Ethylenediamine tetraacetate
(b) Table 3 shows the effects of metal ions.

[Table] 8 Isoelectric point around PH5.7 (ampholine isoelectric focusing method). 9 Molecular weight: 160,000 (monomer), but polymerized to 320,000
(Sephadex G-200 gel filtration method). The molecular weight of 10 subunits is 80,000 (SDS disk electrophoresis method). 11 Crystal form Hexagonal shape (pink color). 12 Prosthetic group Copper ion Although agmatine oxidase, which has the above-mentioned physicochemical properties, is a completely new enzyme, it is considered to be classified as a diamine oxidase when classified based on substrate specificity. To date, putrescine oxidase is the only known diamine oxidase of microbial origin. In addition, diamine oxidase from pig kidney is known as an animal, and diamine oxidase from the Fabaceae family is known as a plant. Also, Aspergillus
terreus amine oxidase belongs to the same type as animal or plant diamine oxidase. The properties of these amine oxidases and agmatine oxidases are compared and shown in Table 4.
However, putrescine oxidase is used in the Agricultural and
Biological Chemistry vol. 30, pp. 1202-1210 (1966), and pig kidney diamine oxidase was described by Y. Yamada et al.
Biochemical and Biophysical Research by
Communications vol. 29, pp. 723-727 (1967), and legume diamine oxidase is described in Biochemical Journal vol. 91, pp. 171-182 (1964) and Method in Enzymology 17 by JMHill et al. Volume B,
It is described on pages 730-735, and Aspergillus
terreus amine oxidase was determined by Yamada et al. (Y.
Agricultural and
Biological Chemistry vol. 29, pp. 864-869 (1965) and Method in Enzymology vol. 17 B,
It is described on pages 705-709.

【table】

【table】

[Table] As is clear from Table 4, agmatine oxidase used in the present invention is different from putrescine oxidase, which is the only diamine oxidase of microbial origin, in terms of substrate specificity, behavior toward inhibitors, molecular weight, prosthetic group, etc. clearly different in this respect. Also, animal origin,
It differs greatly from diamine oxidase of plant origin in its substrate specificity and molecular weight. Therefore, the agmatine oxidase used in the present invention is a completely new diamine oxidase of microbial origin. Agmatine oxidase used in the present invention can be obtained by culturing Penicillium chrysogenum IFO4626, but the medium used is
Any synthetic or natural medium can be used as long as it contains a suitable amount of carbon sources, nitrogen sources, inorganic substances, and other nutrients, and may be either liquid or solid.
Usually a liquid medium is used. and one or two agmatine oxidase inducers such as agmatine, putretsucine, spermidine, and spermine.
More than one type can be used in combination as appropriate. Regarding culture conditions, the pH at the start of culture is usually 4 to 4.
It is carried out in the range of 7, preferably around 5 to 6. The culture temperature is in the range of 20 to 40°C, preferably in the range of 25 to 35°C. If cultured for 12 to 120 hours under such conditions, a significant amount of agmatine oxidase will be produced in the culture. Agmatine oxidase produced and accumulated in the culture in this way is collected by the following method. Since agmatine oxidase is mainly present in bacterial cells, the bacterial cells are collected by filtration after culturing, thoroughly washed with water or buffer, suspended in an appropriate amount of buffer, and the agmatine oxidase in the bacterial cells is collected. Extract. In this case, extraction can be performed by a conventional enzyme isolation method. On the other hand, agmatine oxidase released from the bacterial cells into the culture solution can also be collected from culture filtration by a conventional method.
To further purify the crude agmatine oxidase obtained from these bacterial cell extracts or culture fluids, methods such as isoelectric precipitation, ion exchange chromatography, fractional precipitation with ammonium sulfate, column chromatography with hydyloxyapatite, etc. Methods such as gel filtration using Sephadex and affinity chromatography may be appropriately combined or repeated, and other purification means may be used as necessary. The highly purified agmatine oxidase-containing solution thus obtained is single in disk electrophoresis analysis, and by further concentrating and crystallizing with ammonium sulfate, hexagonal plate-shaped crystals can be obtained. Next, a method for measuring the activity of agmatine oxidase used in the present invention will be described. 4-aminoantipyrine 10mg, phenol 0.2
Prepare a coloring reagent by dissolving 10 mg of peroxidase in 100 ml of 0.2 M phosphate buffer (PH7.0).
Agmatine (10mM) 0.5ml to 1.5ml of this coloring reagent
Add 0.5 ml of the enzyme solution and react, and measure the change in absorbance at 505 nm per minute. The agmatine oxidase activity unit of the enzyme solution is calculated as follows. That is, the amount of agmatine oxidase that produces 1.0 nmol of hydrogen peroxide per minute is defined as 1 unit. One unit of agmatine oxidase corresponds to an increase in absorption of 0.0025 per minute at 505 nm. Next, a method for quantifying agmatine using the novel enzyme agmatine oxidase of the present invention will be explained.
Methods for quantifying agmatine include (a) a method of allowing agmatine oxidase to act on agmatine in the presence of oxygen and quantifying the hydrogen peroxide produced, (b) a method of quantifying the ammonia produced as well, and (c) a method of quantifying the ammonia produced as well. By reacting 3-methyl-2-benzothiazolinone hydrazone with γ-guanidinobutyraldehyde and colorimetrically determining the absorbance at 670 nm,
Method for quantifying agmatine [quantification of aldehyde is
MAPaz (Archives of Biochemistry and
Biophizics 109 Vol. 548-559 (1965)] and (d) a method in which agmatine oxidase is allowed to act on agmatine in the presence of oxygen and the amount of oxygen absorbed by this system is measured. Here, we will describe the method (a) of quantifying agmatine by measuring the amount of hydrogen peroxide produced. That is, in the activity assay section, the concentration of agmatine was 20 μM, 40 μM, 60 μM,
By changing the concentration to 80 μM, 100 μM, 120 μM, and 160 μM, and adding about 3000 units of enzyme with a specific activity of about 5000, the reaction was carried out for 20 minutes and the absorption value of the reaction at 505 nm was determined, and the following results were obtained. .

[Table] That is, substrate concentration (agmatine concentration) and 505nm
A linear relationship is observed with the absorbance value of the reaction solution at . This principle allows the quantification of unknown concentrations of agmatine in solution. Furthermore, it has become possible to measure the agmatine concentration in the solution using agmatine oxidase. This fact suggests the creation of new quantitative means and kits for the field of agmatine quantification. Next, manufacturing examples and examples of the present invention will be described. Production example Penicillium chrysogenum IFO4626, medium composition glucose 0.1%, spermidine 0.025%,
_{KH2PO4 0.1} %, _K2HPO4 0.15 _% , _MgSO4・
Inoculate medium 9 consisting of 0.02% _7H2O and incubate at 28℃.
Incubate for 48 hours. The seed culture solution thus obtained was mixed with putretsucine 0.25%, KH ₂ PO ₄ 0.1%, K ₂ HPO ₄ 0.15
%, _{MgSO4.7H2O0.02} % ₍ before sterilization)
In addition to pH 5.5) 80, perform main culture at 28°C for 48 hours. After culturing, collect the bacterial cells by filtration (approximately 420
g), 0.02% mercaptoethanol and 0.1mM phosphate buffer (PH7.0) (all phosphate buffers include
0.02% mercaptoethanol and 0.1mM EDTA
(contains), suspend in the same buffer, and then crush with a Dyno Mill. Supernatant fraction 3.2 was obtained from this crushed solution by centrifugation (7000 rpm, 20 minutes). The extract was immediately diluted with DEAE-equilibrated with the above buffer.
Pass through cellulose column 1.2. With this operation, agmatine oxidase is adsorbed. Wash unadsorbed impure proteins with the same buffer, then increase the buffer concentration to 0.2M to elute agmatine oxidase. The eluted active fraction is subjected to ammonium sulfate fractionation at an ammonium sulfate concentration of 45% saturation, and then dialyzed against 0.01M phosphate buffer (PH7.0). The dialyzed enzyme is passed through a DEAE-cellulose column equilibrated with 0.01M phosphate buffer (PH 7.0). After washing with the same buffer, 0.01M
The enzyme is separated by linear concentration gradient method using ~0.2M phosphate buffer (PH7.0). Active fraction is 35%~45
Fractionate ammonium sulfate at a concentration of % saturation and dialyze against 0.01M phosphate buffer (PH7.0). The dialyzed enzyme solution is passed through a hydroxyapatite column equilibrated with the same buffer solution for adsorption. Unadsorbed proteins are washed with the same buffer, and the enzyme is eluted using a linear concentration gradient method of 0.01-0.1M phosphate buffer (PH7.0). The eluate was concentrated with 45% saturated ammonium sulfate and then purified with Sephadex G-
Perform a molecular comb with 200 ml. The active fraction of agmatine oxidase contained in the flowthrough is concentrated and crystallized by adding ammonium sulfate. The crystals obtained are hexagonal in shape. The specific activity of the purified enzyme is approximately 640 times higher than that of the cell extract, and the activity ratio is approximately 30%. Example 1 (a) Reagents used (1) Test solution Agmatine-containing solution (concentration unknown) 0.5
ml (2) 1.5ml of color reagent consisting of the following composition: 10mg in 100ml of 0.2M phosphate buffer (PH7.0)
Contains 4-aminoantipyrine, 5 mg peroxidase (specific activity 1000), and 0.2 ml phenol. (3) Agmatine oxidase (6000unit/ml)
Put 0.5ml of the above (1) and (2) into a test tube and preheat at 35℃ for 3 minutes. Next, add the enzyme solution and react at 35°C for 20 minutes. On the other hand, as a control, a sample using water instead of the test liquid was treated in the same manner. of the test liquid
The absorption value at 505 nm was determined, and the difference ΔA from the control was 0.235. Based on the calibration curve shown in FIG. 5, the agmatine content in the test solution was analyzed to be 37.6 μM, or 94 nmol. Example 2 Enzyme solution: Dissolve agmatine oxidase (3000 units/ml) in 0.2M phosphate buffer (PH7.0) Add 100μ of the test solution (1) in Example 1 to 1 ml of the enzyme solution.
was added and reacted for 20 minutes at 30°C, and the amount of enzyme consumed due to enzyme activity was measured using an enzyme electrode. On the other hand, as a control, a sample using water instead of the test liquid was treated in the same manner. Separately, a calibration curve was created in the same manner using an agmatine solution of known concentration instead of the test solution. As a result, the agmatine content of the test solution was 35 μM. Example 3 Enzyme solution: Dissolve agmatine oxidase (6000 units/ml) in 0.1M phosphate buffer (PH7.0) Enzyme solution containing glutamate dehydrogenase: α- in 0.1M phosphate buffer (PH8.0) Dissolve ketoglutaric acid (15mM), NADPH (0.4mM), and glutamic acid dehydrogenase (15unit/ml) 1ml of enzyme solution in 100μ of test solution (1) of Example 1 above
was added and allowed to react at 37°C for 20 minutes. Then, 2 ml of glutamate dehydrogenase-containing enzyme was added and 37
After reacting at ℃ for 5 minutes, absorbance at 340 nm was measured. On the other hand, as a control, water was used instead of the test liquid and treated in the same manner. Separately, a calibration curve was prepared in the same manner using an agmatine solution of known concentration instead of the test solution. As a result, the agmatine content of the test solution was 36 μM.

[Brief explanation of the drawing]

Figure 1 shows the PH activity curve of agmatine oxidase that can be used in the present invention, Figure 2 also shows the PH stability, Figure 3 shows the optimal temperature, and Figure 4 shows the temperature stability. It is something. FIG. 5 is a graphical representation of Table 5 for agmatine determination.

Claims (1)

[Claims] 1. A method for quantifying agmatine, which comprises allowing agmatine oxidase to act on agmatine or a substance containing it in the presence of oxygen, and measuring the amount of oxygen consumed, or quantifying ammonia, aldehyde, or hydrogen peroxide produced.