JPH0218833B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an enzyme that oxidizes agmatine (hereinafter referred to as agmatine oxidase). More specifically, the present invention relates to 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 discovered that this enzyme is a new enzyme and named it 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 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. Penicillium chrysogenum IFO 4626, which can grow using spermine, spermidine, putretsucine, agmatine, etc. as a single carbon and nitrogen source, was cultured in a medium containing spermine, spermidine, putretzine, agmatine, etc. discovered that a novel agmatine oxidase was produced and accumulated in culture, named it agmatine oxidase, and completed the present invention. The agmatine oxidase obtained in the present invention is
It is a novel enzyme that has substrate specificity for agmatine, slightly acts on other diamines, but does not substantially act on polyamines. The agmatine oxidase of the present invention has the following physicochemical properties. 1 Action: As shown in the following formula, it acts on agmatine and converts 1 mol of agmatine into 1 mol of γ-guanidinobutyraldehyde, 1 mol of ammonia, and 1 mol of ammonia.
Produces moles of hydrogen peroxide. b) Confirmation of production of hydrogen peroxide When agmatine oxidase is allowed to act on agmatine in the presence of oxygen, and then peroxidase, 4-aminoantipyrine, and phenol are added to the enzyme system and allowed to react, quinone imine dye is produced in the reaction system. (The reaction between hydrogen peroxide, peroxidase, 4-aminoantipyrine, and phenol is shown in Clin. Chem. vol. 20, p. 470 (1974)). b) Confirmation of the production of ammonia When agmatine is reacted with agmatine oxidase in the presence of oxygen, and then sodium hypochlorite, phenol, sodium hydroxide, and sodium nitroprusside are added to the enzyme system and reacted, the reaction solution system contains ammonia. Phenol is produced (For the reaction of ammonia with sodium hypochlorite, phenol, sodium hydroxide, and sodium nitroprusside, see J.Clin.Path.Volume 13, 156)
(1960)). c) Confirmation of production of γ-guanidinobutyraldehyde Agmatine oxidase is allowed to act on agmatine in the presence of oxygen, and then potassium permanganate is 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 are solvent system 1 [0.1 molar phosphate buffer PH7.0] and solvent system 2 [butanol:acetic acid:water = 4:1:5 (volume ratio)]. It is. After development, ninhydrin reaction,
The Sakaguchi reaction was performed and it was confirmed that the Rf value and color tone matched those of the standard product. 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 measured by the peroxidase, phenol, and 4-aminoantipyrine method for hydrogen peroxide produced. 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% activity remains even after treatment at 40°C for 20 minutes at pH 7.0 (as shown in Figure 4). 7 Effects of inhibitors and metal ions a) Table 2 shows the effects of various inhibitors.

【table】

[Table] *Parachloromercurybenzoate **Ethylenediaminetetraacetate 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 may reach 320,000 when polymerized (Sephadex G-200 gel filtration method). The molecular weight of 10 subunits is 80,000 (SDS disk electrophoresis method). 11 Crystal form Hexagonal plate 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 H. Yamada et al.
Biochemical and Biophysical Besearch by
Communications vol. 29, pp. 723-727 (1967), and leguminous diamine oxidase is described in J. M. M. Hill et al.
Biochemical Journal Vol. 91, pp. 171-182 (1964) and Method in Enzymology Vol. 17 B,
It is described on pages 730-735, and Aspergillus
terreus amine oxidase was determined by Yamada et al. (H.
Agricultural and
Biological Chemistsy Vol. 29, pp. 864-869 (1965)
and Method in Enzymology Volume 17 B, 705~
It is described on page 709.

【table】

[Table] As is clear from Table 4, the agmatine oxidase of 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. Furthermore, diamine oxidases of animal origin and plant origin differ greatly in their substrate specificity and molecular weight. Therefore, the agmatine oxidase of the present invention is a completely new diamine oxidase of microbial origin. The agmatine oxidase of the present invention can be obtained by culturing Penicillium chrysogenum IFO4626, but the medium to be used may be a synthetic medium or a natural medium as long as it contains moderate amounts of carbon sources, nitrogen sources, inorganic substances, and other nutrients. Although either liquid or solid media can be used, liquid media are usually used. It is also possible to use one type or a combination of two or more of agmatine oxidase inducers such as agmatine, putrescine, spermidine, and spermine. Regarding culture conditions, the pH at the start of culture is usually 4 to 4.
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 under such conditions for 12 to 120 hours, 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 the bacterial cells, the bacterial cells are collected using an appropriate method after the culture is completed, thoroughly washed with water or a buffer solution, and suspended in an appropriate amount of buffer solution. 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 the culture using a conventional method.
In order 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 hydroxyapatite, and sepadex Methods such as gel filtration and affinity chromatography may be appropriately combined or repeated, and other conventional purification methods may be used as necessary. The highly purified agmatine oxidase-containing solution obtained in this way 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 color 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 the above-mentioned 505 nm. Next, a method for quantifying agmatine using the novel enzyme agmatine oxidase provided by the present invention will be described. For the determination of agmatine,
(b) A method for quantifying hydrogen peroxide produced by agmatine oxidase acting on agmatine in the presence of oxygen, (b) A method for quantifying ammonia produced in the same way, (c) A method for quantifying ammonia produced in the same way, (c) A method for quantifying the produced ammonia in the same way, and (c) a method for quantifying the produced hydrogen peroxide in the presence of oxygen. A method for quantifying agmatine by colorimetrically measuring the absorbance at 670 nm with the action of 3-methyl-2-benzothiazolinone hydrazone.
Biochemistry and Biophysics Volume 109 548-559
(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 section of activity measurement method, the concentration of agmatine is 20 μM, 40 μM, 60 μM, 80 μM, 100 μM,
By changing the concentrations to 120 μM and 160 μM, adding about 3000 units of an enzyme with a specific activity of about 5000, and carrying out a reaction for 20 minutes, the absorption value of the reaction solution at 505 nm was determined, and the following results were obtained.

[Table] That is, a linear relationship is observed between the substrate concentration (agmatine concentration) and the absorbance value of the reaction solution at 505 nm.
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, examples of the present invention will be described. Example 1 Penicillium chrysogenum IFO4626 was grown in a medium containing glucose 0.1%, spermidine 0.025%,
KH ₂ PO ₄ 0.1%, K ₂ HPO ₄ 0.15%, MgSO ₄・
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 straining (approximately 420
g), 0.02% mercaptoethanol and 0.1mM
Wash with 0.01M phosphate buffer (PH7.0) containing EDTA (all phosphate buffers hereinafter contain 0.02% mercaptoethanol and 0.1mM EDTA), suspend in the same buffer, and crush with a Dyno Mill. . The supernatant fraction is extracted from this crushed solution by centrifugation (7000 rpm, 20 minutes).
Got 3.2. The extract is immediately passed through a DEAE-cellulose column 1.2 equilibrated with the above buffer. 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 (PH7.0). After washing with the same buffer, add 0.01M to 0.2M phosphate buffer (PH
Elute the enzyme using the linear concentration gradient method according to 7.0). The active fraction is subjected to ammonium sulfate fractionation at an ammonium sulfate concentration of 35% to 45% saturation, and then dialyzed 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. Wash the unadsorbed protein with the same buffer, and remove the enzyme.
Elute with 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 subjected to molecular sieving using Sephadex G-200. The active fraction of agmatine oxidase contained in the flowthrough is concentrated and crystallized by adding ammonium sulfate. The obtained crystals have a hexagonal plate shape. The specific activity of the purified enzyme is approximately 640 times higher than that of the cell extract.
The yield of activity is approximately 30%. Reference Example A) Reagents used (1) Test solution containing agmatine (concentration unknown) 0.5 ml. (2) Coloring reagent consisting of the following composition: 1.5ml 10mg4 in 100ml of 0.2M phosphate buffer (PH7.0)
-Contains aminoantipyrine, 5mg peroxidase (specific activity 1000), and 0.2ml phenol. (3) Enzyme (6000 units/ml) 0.5 ml Place (1) and (2) above into a test tube and preheat at 35°C 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
Obtain the absorption value at 505 nm, and compare the difference with the control △
A 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.

[Brief explanation of drawings]

Figure 1 shows the pH of agmatine oxidase of the present invention.
These are activity curves; Figure 2 shows the PH stability, Figure 3 shows the optimum temperature, and Figure 4 shows the temperature stability. FIG. 5 is a graphical representation of Table 5 for agmatine determination.

Claims (1)

[Scope of Claims] 1. Agmatine oxidase characterized by having at least the following physical and chemical properties. a Action: Acts on agmatine, producing 1 mole of γ-guanidinobutyraldehyde and 1 mole of agmatine.
Produces 1 mole of ammonia and 1 mole of hydrogen peroxide. b Substrate specificity It has specificity for agmatine, slightly acts on other diamines, but does not substantially act on polyamines. c Optimum pH around 6.5-7.0. d Stable PH range 5.5-7.5 (40℃, 20 minutes). e Molecular weight 160000 (gel filtration method).