JPH0147151B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing ethanolamine oxidase. Until now, only bacteria belonging to the genus Arthrobacter were known as ethanolamine oxidase-producing bacteria [J. Biol.
chem., 239 , (1964) 2189]. The present inventors have discovered that bacteria B-0783 belonging to the genus Bacillus collected from the soil of an orchard in Shimohata, Ohito-cho, Tagata-gun, Shizuoka Prefecture have substrate specificity for at least ethanolamine in their culture. , and has the action of catalyzing a reaction that consumes 1 mol of oxygen and water per 1 mol of ethanolamine and produces 1 mol of glycolaldehyde, 1 mol of ammonia, and 1 mol of hydrogen peroxide. discovered that it produces oxidase. First, the characteristics of the culture of the above-mentioned bacterium B-0783 on various media based on macroscopic and microscopic observations are as follows.
It is as described below. (A) Macroscopic observation (1) Broth agar plate medium (30°C, 18 hours) Forms round, flat colonies, with smooth grey-white to pale yellowish-gray surroundings. No soluble pigments are produced. (2) Broth agar slant medium (30°C, 18 hours) Grows well in a linear pattern and is grayish-white to light yellowish-gray in color. No soluble pigments are produced. (3) Meat juice liquid medium (30℃, 32 hours) Growth is rather weak. Produces precipitation. (B) Microscopic observation After culturing on ordinary agar medium at 30°C for 18 hours, the morphological characteristics were observed. (1) Cell shape and size Single, double, or short-chain rod-shaped bacteria with rounded ends, measuring 1.0 to 1.5 x 2.0 to 5.0 μm. (2) Presence or absence of polymorphism None. (3) Motility and flagella Moves with pericilla. (4) Spores (shape, position, size) Spores are oval or oval and are formed at or near the edge of the bacterial body, and the bacterial body becomes spindle-shaped due to the spores. (C) Physiological properties Nitrate reduction + denitrification reaction − MR test − VP test − Production of indole − Production of hydrogen sulfide − Hydrolysis of gelatin + (weak) Hydrolysis of starch − Utilization of citric acid (Simmons medium) − (Christenzen medium) − Utilization of nitrate + Utilization of ammonium salt − Production of pigment − Oxidase + Calatase + Urease (SSR medium) − (Christenzen medium) − Growth range (PH) Optimal PH 6.5 to 8.0 Growth PH 5.0 ~9.0 (Temperature) Optimum temperature 25-37℃ Growth temperature 10-42℃ Attitude toward oxygen Aerobic OF test (Hugh Leifson medium)
Al (becomes alkaline) OF test (denatured medium) Al (becomes alkaline) Decomposition of esculin - Decomposition of cellulose - Gram staining + acid-fast staining - Acid, gas production Acid Gas Adonitol - - L(+)arabinose - - Cellobiose − − Dulcitol − − Mesoerythol + − D(−)Fructose − − Fucose − − D(+) Galactose − − D(+) Glucose − − Glycerin − − Inositol − − Inulin − − Lactose − − Maltose − - D-mannitol - - D-mannose - - melezitose - - melibiose - - raffinose - - L (+) rhamnose - - salicin - - L-sorbose - - sorbitol - - starch - - satucalose - - trehalose - - D-xylose - - DNA GC content (Tm method) 37.3% Based on the above mycological properties, this bacterium B-0783 is Gram-positive and has the characteristics of being a spore-forming bacillus. Of Determinative Bacteriology (Bergey,
s Manual of Determinative Bacteriology)
According to the 8th edition (1974), this strain was recognized as belonging to the genus Bacillus. Furthermore, the properties of this strain were described in the Burgess Manual.
Of Determinative Bacteriology No. 7
edition, 8th edition, and The Genus Bacillus (Agriculture Handbook,
427), but there were no descriptions of bacterial species that closely matched the characteristics of this strain, so this strain was compared to Bacillus.
It was identified and named Bacillus sp.B-0783. Furthermore, this strain was deposited at the Institute of Microbiology, Agency of Industrial Science and Technology as "FERM-P No. 5798 (FERM-P No. 5798) and FERM-P No. 362 (FERM BP-362)". The present invention was completed based on the above findings, and is characterized by culturing ethanolamine oxidase-producing bacteria belonging to the genus Bacillus in a medium and collecting ethanolamine oxidase from the culture. It is a manufacturing method. As the bacteria used in the present invention, the above-mentioned Bacillus sp. B-0783 is one example, and
In addition to this bacterium, other bacteria belonging to the genus Bacillus that produce ethanolamine oxidase include
All can be used in the present invention. In carrying out the present invention, ethanolamine oxidase-producing bacteria belonging to the genus Bacillus are cultured by a conventional method for producing enzymes. The form of culture may be liquid culture or solid culture, and from an industrial perspective, it is advantageous to inoculate cells of ethanolamine oxidase-producing bacteria into a production medium and perform deep aeration agitation culture. As the nutrient source for the medium, a wide variety of nutrients commonly used for culturing microorganisms can be used. The carbon source may be any assimilable carbon compound, such as glucose, sucrose, lactose, maltose, starch, dextrin, molasses, waste molasses, and glycerin. The nitrogen source may be any available nitrogen compound, such as corn steep liquor, soybean flour, cottonseed flour, wheat gluten, peptone, meat extract, yeast extract, casein hydrolyzate, and the like. In addition, salts such as phosphate, magnesium, calcium, potassium, sodium, zinc, iron, manganese, and halogen are used as necessary. Furthermore, in order to induce the production of ethanolamine oxidase in the medium, it is preferable to add about 0.1 to 1% of an alkylamine such as ethanolamine or propulamine to the medium as an inducer. The culture temperature can be changed as appropriate within a range that allows the bacteria to grow and produce ethanolamine oxidase, but is preferably 26 to 33°C, particularly about 30°C. Cultivation time varies slightly depending on conditions, but usually it is sufficient to culture for about 15 to 25 hours, and even more for 200 to 25 hours.
Aeration may be performed while stirring at 400 rpm, and the culture may be terminated at an appropriate time, taking into account the time when ethanolamine oxidase reaches its maximum titer. In the thus obtained culture of ethanolamine oxidase-producing bacteria, ethanolamine oxidase is mainly contained within the cells. For example, when collecting this enzyme, the cells of the obtained culture are first collected by means such as filtration or centrifugation, and then the cells are subjected to ultrasonic treatment, French press treatment, or glass bead treatment. Destroy by mechanical destruction means such as lysozyme or enzymatic destruction means such as lysozyme, and if necessary, surfactant such as Triton Agents may also be added. The ethanolamine oxidase-containing solution thus obtained is then concentrated, or without concentration, salted out using a soluble salt, such as ammonium sulfate, or dissolved in a hydrophilic organic solvent, such as methanol, ethanol, acetone. The enzyme may be precipitated using , isopropanol, or the like. Furthermore, after dissolving this precipitate in water or a buffer solution, it is dialyzed with a semi-permeable membrane as necessary, and then DEAE-Sephadex, DEAE-
Chromatography using ion exchange resins such as Cepharose, DEAE-cellulose, carboxymethyl-cellulose, carboxymethyl-Sepharose, carboxymethyl-Sephadex, Cephadex G200, Cepharose CL-
The purified ethanolamine oxidase is purified by chromatography using a gelling agent such as a molecular sieve such as 6B or Cephacryl S-200, and then subjected to a process such as freeze-drying to obtain purified ethanolamine oxidase. Next, the method for measuring the activity and properties of ethanolamine oxidase of the present invention will be described. Activity measurement method 0.2M Tris-HCl buffer (PH8.0) 0.1ml 0.3% 4-aminoantipyrine 0.02ml 3mM N,N-diethyl-m-toluidine 0.05ml (45U/ml) Peroxidase 0.05ml 0.1M Ethanolamine 0.05 ml distilled water 0.18ml total 0.45ml 0.45ml of the reaction solution having each of the above compositions was heated at 37℃ for 2 hours.
Preheat for 1 minute, then add 50μ of the enzyme solution and react at 37°C for 10 minutes. The reaction is stopped by adding 2.5 ml of ethanol, and the color development is then measured by absorbance at a wavelength of 550 nm. Enzyme activity is defined as 1 unit (1U), which is the activity that produces 1 μmole of hydrogen peroxide per minute. Calculation of titer follows the following formula. Enzyme activity (U/ml) = △A ₅₅₀ × _0.25 It has the function of catalyzing a reaction that consumes water and produces 1 mole of glycolaldehyde, ammonia and hydrogen peroxide. H ₂ N−CH ₂ CH ₂ −OH+O ₂ +H ₂ O Ethanolamine → OHC−CH ₂ −OH+NH ₃ +H ₂ O ₂ Glycolaldehyde Substrate specificity In the above activity measurement method, the following various The activity was measured using the substrate. The results were as shown in Table 1.

[Table] Optimal PH In the buffer solution used in the activity measurement method described above, the PH
The enzyme activity was measured using a phosphate buffer with pH 6 to 7 and a Tris-hydrochloric acid buffer with pH 7 to 9. As a result, as shown in Figure 1, this enzyme has a pH of 7 to 7.5.
It is recognized that the optimum pH is within the range of . PH stability 2.5U/ml enzyme solution was mixed with 100mM various buffers (dimethylglutarate buffer with pH 5-7.5, pH 8-7.5).
After heat treatment at 60° C. for 15 minutes with Tris-hydrochloric acid buffer (pH 9.5, glycine-sodium hydroxide buffer (PH 9-10)), the residual activity was measured based on the activity measurement method described above. As a result, as shown in Figure 2,
This enzyme was found to be stable within the pH range of 6 to 8. Thermostability After heat-treating a 2.5U/ml enzyme solution with 10mM Tris-HCl buffer (PH8.0) at 40-75℃ for 10 minutes, its residual activity was measured based on the activity measurement method described above. did. As a result, as shown in Figure 3, this enzyme was found to be stable up to 60°C. Isoelectric point around PH4.60 (measured by focusing electrophoresis using carrier ampholite)

【table】

[Table] Molecular weight Molecular weight; 320000±30000 (Sepharose CL-6B
By gel filtration method using column size: 2.5
×90cm, solvent: 10mM Tris-HCl buffer, PH
7.5, including 0.5MKCl) Based on the above properties of this enzyme, this enzyme is recognized as ethanolamine oxidase. Furthermore, this ethanolamine oxidase
Used for quantifying samples that release ethanolamine or samples that contain ethanolamine. For example, by allowing phospholipase D to act on a phospholipid mixture such as lecithin, sphingomyelin, lysolecithin, and phosphatidylethanolamine, and allowing this ethanolamine oxidase to act on the ethanolamine produced, the enzymatic action of ethanolamine oxidase By measuring the enzyme consumed or the hydrogen peroxide, ammonia, or ammonium ion produced, it is possible to specifically quantify only phosphatidylethanolamine in the phospholipid mixture. In addition, the measurement may be carried out by electrical means using an electrode sensitive to the target component, such as an oxygen electrode, a hydrogen peroxide electrode, an ammonia electrode, or an ion electrode. The immobilized enzyme may be used as an enzyme electrode fixed to an electrode surface used for electrical means. Furthermore, when measuring hydrogen peroxide, colorimetry is preferably performed using a system using one or more coloring agents that undergo a color change in the presence of hydrogen peroxide. For example, the amount of hydrogen peroxide produced is determined by the strength of the color using xylenol orange and a tetravalent titanium compound that reacts with the produced hydrogen peroxide to form a stable red color. Examples include a method of measuring a change in color tone by a reaction with phenol, 4-aminoantipyrine, and peroxidase. Also, instead of this 4-aminoantipyrine, 4
- Aminofenazone may be used. Furthermore, using a reagent containing diethyl-m-toluidine, 4-aminoantipyrine, and peroxidase,
The amount of hydrogen peroxide produced may be measured by the intensity of its coloration. Other methods include quantitative measurement of hydrogen peroxide using a combination of 2,6-dichlorophenol indophenol and peroxidase, quantitative measurement of hydrogen peroxide using a combination of guaiac butter and peroxidase, and quantitative measurement of hydrogen peroxide using a combination of homovanillic acid and peroxidase. Quantitative methods using peroxidase, such as measurement, are preferred because they are simple and accurate. Furthermore, this reagent for quantifying hydrogen peroxide may be made into a simple laminated integral type by coating it on the surface of a carrier such as a film or paper. Next, the present invention will be specifically described with reference to Examples, but the present invention is not limited thereto. Example 1 Add 20 liters of medium (peptone 1.5%, powdered yeast extract 0.5%, Kcl
0.2%, NaCl 0.1%, _{K2HPO4 0.1} %, MgSo4 _0.1
%, silicone KM-72 0.05%, ethanolamine 0.5%, PH6.8), sterilized at 120℃ for 20 minutes, then adjusted the temperature to 30℃, and precultured Bacillus sp. with the same medium composition. B-0783 culture solution 500
ml (rotary shaker, 30℃, 20 hours culture), stir at 300 rpm, and aerate at 20/min.
Cultured for 17 hours. After incubation, the culture was incubated at 5000 rpm, 20
The cells were collected by centrifugation for 1 minute. Next, this bacterial cell was treated with 500mg lysozyme (manufactured by Eisai) in Step 2.
After suspending in 10nM phosphate buffer (PH7.0) containing
The mixture was heated at 37°C for 2 hours to obtain 1700ml of crude enzyme solution (0.2U/ml). Next, 850 ml of cold acetone was added to this, and the precipitated insoluble matter was centrifuged (5000 rpm,
10 minutes). Furthermore, 1200 ml of cold acetone was added to this supernatant to precipitate it.
The precipitate was collected by centrifugation at 5000 rpm for 10 minutes at 0°C. This precipitate containing 0.5M KCl
After dissolving in 100ml of 0.1mM Tris-HCl buffer (PH8.0), this was desalted by dialysis using a semipermeable cellulose tube, and then freeze-dried to obtain an ethanolamine oxidase-containing powder (0.1U/mg,
3.2g) was obtained. Furthermore, this ethanolamine oxidase-containing powder may be purified as in Example 2 below. Example 2 After dissolving 1.0 g of the powder obtained in Example 1 in 15 ml of 10 mM Tris-HCl buffer (PH8.0), DEAE-Sepharose CL was equilibrated with the same buffer.
-6B (manufactured by Pharmacia) column (2.5 x 21 cm)
It was charged and adsorbed, and washed with 500 ml of the same buffer solution. After this, add 200ml of the same buffer and 200ml of
The protein was eluted by changing the salt concentration using the same buffer containing 0.5M KCl. 1 fraction
The active fractions of fractions No. 56 to 66 were collected and combined, and then dialyzed against 10 mM Tris-HCl buffer (PH8.0), and then lyophilized to ethanol. Amine oxidase powder (0.7 U/mg, 110 mg) was obtained. Example 3 The same procedure as in Example 1 was carried out using a medium containing 0.5% propylamine instead of ethanolamine in the medium composition of Example 1, and ethanolamine oxidase-containing powder 0.09 U/mg, 3.0
I got g.

[Brief explanation of drawings]

FIG. 1 shows the optimum PH curve for the ethanolamine oxidase of the present invention, FIG. 2 shows the PH stability curve of the ethanolamine oxidase of the present invention, and FIG. 3 shows the thermostability curve of the ethanolamine oxidase of the present invention.

Claims (1)

[Scope of Claims] 1. A method for producing ethanolamine oxidase, which comprises culturing ethanolamine oxidase-producing bacteria belonging to the genus Bacillus in a medium, and collecting ethanolamine oxidase from the culture. 2. The method for producing ethanolamine oxidase according to claim 1, wherein the medium is a medium to which an alkylamine is added as an inducer. 3. The method for producing ethanolamine oxidase according to claim 2, wherein the alkylamine is ethanolamine or propylamine. 4 Ethanolamine oxidase-producing bacteria belonging to the genus Bacillus are Bacillus sp.
A method for producing an ethanolamine oxidase-producing bacterium according to claim 1, 2 or 3, which is a 0783 bacterium.