JPS632592B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides novel L-arginine oxidase
(L-Arginine oxydase), and its production method,
Regarding the producing bacteria. Conventionally, amino acids of different types had the same effect on each other.
As an oxidase, L-amino acid oxidase
was known [J.Biol.Chem.,153, 387
(1944), J. Bacteriol.55, 401 (1948),
Biochem.J.,50, 258 (1952), J. Bacteriol.121,
656 (1975), Agric.Biol.Chem.432531 (1979),
Method in Enzymology B (1971) etc.]. These L-amino acid oxidases
Has wide quality specificity and acts strongly on one type of amino acid
For example, snake venom L-amino acid oxidizer
The enzymes are L-leucine, L-methionine, and L-phene.
It acted to the same extent as nilalanine. The present inventors have investigated the problem of removing sludge from irrigation canals in Nagaoka City, Niigata Prefecture.
L-
Has strong substrate properties for arginine and reacts with the following
formula〔〕 It has an enzymatic action that strongly catalyzes the reaction shown in
The enzyme was discovered and its substrate specificity and
As is clear from its enzymatic action, the known L-
It is a completely different enzyme from amino acid oxidase.
Therefore, it was recognized as a new enzyme and L-arginine oxidase.
It was named ``Ze''. First, the activity of L-arginine oxidase of the present invention
Describes the assay method, substrate specificity, and enzymatic action.
Ru. (1) Activity measurement method 0.2ML - Arginine solution 0.1ml, 0.2M dimethylene
Luglutaric acid-sodium hydroxide buffer (PH
6.5) 0.2ml, 0.3% 4-aminoantipyrine 0.1
ml, 0.2% phenol 0.1ml, peroxidase
(50U/ml) and 0.3ml of distilled water.
Dispense 0.9ml of the reaction solution into small test tubes and incubate at 37℃ for 3 minutes.
After prewarming for a while, add 0.1ml of enzyme solution and
Incubate at 37°C for 10 minutes, then add 2.0 ml of ether.
The reaction was stopped by adding alcohol, then the
The reaction solution was measured colorimetrically at 480nm, and its absorbance
seek. As an enzyme unit, 1 μmol per minute at 37℃
The amount of enzyme that produces hydrogen peroxide is 1 unit (1U)
And so. The enzyme titer was calculated according to the following formula. Titer (U/ml) = △A480nmX0.699 ×Dilution ratio (△A480nm indicates the absorbance at 480nm.
vinegar) (2) Substrate specificity Liquid containing L-arginine oxidase 0.047U
0.1ml, 5mM substrate (each substrate listed in Table 1)
Contains 0.1ml of liquid, 1mMEDTA0.1ml, 0.2M dimethycin
Luglutaric acid-sodium hydroxide buffer (PH
6.5) 0.2ml, 0.3% 4-aminoantipyrine 0.1
ml, peroxidase (50U/ml) 0.1ml, 0.2
% phenol 0.1ml, distilled water 0.2ml reaction
The following reaction is based on the activity measurement method.
As a result of measuring the activity against each substrate using
It was as shown in the table.

【table】

[Table] As shown in Table 1 above, the enzyme of the present invention is L
- Strongly acts on arginine, L-lysine, L-aspartic acid, L-alanine, DL
-Exhibits a very weak effect on tryptophan substrates, and also on other D-, L- and
DL-amino acids and L-leucyl-L-lysine, L-leucyl-glycine, L-leucyl-
Since it has no effect on L-arginine, the enzyme of the present invention is recognized to have substrate specificity for L-arginine. (3) Enzyme action Using a reaction system consisting of 0.1ml of 0.5mM arginine solution, 0.9ml of 0.2M dimethylglutaric acid-sodium hydroxide buffer (PH6.5), and 20μl (1.3U) of L-arginine oxidase solution,
The reaction was carried out at 37°C for 10 minutes, and the amount of oxygen consumed and the amount of ammonia produced during the reaction were measured using an oxygen electrode and an ammonia electrode, respectively. As a result, per 0.05μmol of L-arginine, the amount of oxygen consumed was 0.047μmol, and the amount of ammonia produced was
It was 0.048μmol. Use 0.1ml of 0.5mM L-arginine solution instead of L-arginine solution in the activity measurement method, and use L-arginine oxidase solution.
Using 0.1 ml (1.3 U), the amount of hydrogen peroxide produced in the reaction was measured according to the activity measurement method below. As a result, L-arginine
The amount of hydrogen peroxide produced is 0.049μ per 0.05μmol
It was mole hot. From the above results, the following reaction formula [] It is estimated that it catalyzes the enzymatic reaction shown in As is clear from the substrate specificity and enzymatic action described above, the enzyme of the present invention is recognized as a new enzyme that is different from the conventionally known L-amino acid oxidase, and due to its substrate specificity, it is similar to L-arginine oxidase. I named it. Furthermore, although the L-arginine oxidase of the present invention is not limited, its molecular weight,
Km value, isoelectric point, optimum PH, PH stability, thermal stability,
The optimum temperature, the influence of metal salts and chelating agents, and the influence of cofactors will be described. (1) Molecular weight 140000±14000 (by gel filtration method using Cephacryl S-200) (2) Km value Km=25×10 ^-3 M±2.5×10 ^-3 M (relative to L-arginine) (3 ) Isoelectric point PH4.8±0.4 (by electrophoresis using carrier amphorite) (4) Optimum pH As a buffer solution, dimethyl glutaric acid-sodium hydroxide buffer (PH3.8-7.5) (first In the figure,
〇-〇), phosphate buffer (PH6.0-7.8)
(indicated by ●-● in Figure 1) and Tris-
Hydrochloric acid buffer (PH6.8-8.8) (△-△ in Figure 1)
As shown in Figure 1, the results of measuring the activity of this enzyme at each pH were as follows:
The optimum pH of this enzyme was found to be around 6.5 (at 37°C). (5) PH stability As a buffer solution, dimethyl glutaric acid-sodium hydroxide buffer (PH3.8-7.5) (in Figure 2,
〇-〇), phosphate buffer (PH6.0-7.8)
(indicated by ●-● in Figure 2), Tris-hydrochloric acid buffer (PH6.8-8.8) (indicated by △-△ in Figure 2), and glycine-sodium hydroxide buffer (PH8. 7-9.2) (indicated by ▲-▲ in Fig. 2), and after holding at 37°C for 24 hours, the activity of this enzyme was measured, as shown in Fig. 2.
This enzyme was found to be stable around pH 6.0. (6) Thermal stability The thermal stability results when treated with dimethyl glutaric acid-sodium hydroxide buffer (PH6.5) at each temperature for 10 minutes are shown in Figure 3. The enzyme was found to be stable up to 25°C. (7) Optimal PH Using a reaction solution with the same composition as the reaction solution used in the activity measurement method, the optimum temperature was measured at various temperatures, as shown in Figure 4. The temperature was found to be around 25℃. (8) Effects of metal salts and chelating agents The metal salts and chelating agents listed in Table 2 were added to the reaction solution in the activity measurement method, and their effects were determined. The results were as shown in Table 2.

[Table] (9) Influence of cofactors Each of the cofactors shown in Table 3 was added to the reaction solution in the activity measurement method, and the influence was determined. The results were as shown in Table 3.

[Table] Bacterial strain B-0780, which is a new L-arginine oxidase-producing bacterium of the present invention, was used to collect sludge from irrigation canals in Nagaoka City, Niigata Prefecture, with 0.4% meat extract, 1.0% peptone, 0.3% salt, and 0.1 L-amino acid. % and agar 1.5%, and cultured at 30°C for 30 hours.
It was obtained by sorting out grown colonies exhibiting arginine oxidase activity. The characteristics of the culture of this strain B-0780 on various media based on macroscopic and microscopic observations are as described below. A. Macroscopic observation (1) Ordinary agar slant culture medium Grows in a linear pattern, translucent, pale yellow in color, and growth rate is rather slow. No soluble pigments are produced. (2) Ordinary agar plate medium Forms small round colonies, translucent, and pale yellow in color. No soluble pigments are produced. (3) Bouillon medium Growth is weak, but after uniform turbidity, precipitation occurs. B. Morphological characteristics Young cells (12 hours after culture) are straight or slightly curved rod-shaped, mixed rod-shaped, or club-shaped, with V-shaped, W-shaped, Y-shaped, and U-shaped cell arrangements. be observed. Size is 0.5~1.0×1.5~
3.0μ. Older cells (more than 24 hours in culture) have a short rod-like or spherical shape. There is no motility. It has no spore-forming ability. C Physiological and biochemical properties Gram staining, acid-fast staining - OF test O (oxidation) Gelatin degradation - Starch degradation (+) Casein degradation - Aesculin degradation + Catalase production + Oxidase production (+) Urease production - Indole production − H ₂ S production − VP, MR test − Nitrate ring element + BCP milk Utilization of unchanged citric acid Simmons medium + Christen Gan medium + Production of acids from sugars The following types of sugars produce acids, but xylose produces a weak acid in about 4 days, and other sugars in about 7 days. Does not produce gas. Adonitol, L(+)-arabinose, meso-erythritol, fructose, fucose,
Galactose, glucose, glycerin, mannitol, mannose, melibiose, L(+)
- Sugars that do not produce rhamnose, sorbitol, xylose acid Cellobiose, dulcitol, inositol, inulin, lactose, maltose, melezitose, raffinose, salicin, L-sorbose, starch, sucrose, trehalose Attitude towards oxygen Aerobic inorganic nitrogen source Nitrate - Ammonium salt - Denitrification reaction - Growth temperature range 15-37℃ Growth PH range PH6-9 As mentioned above, the B-0780 strain of this bacterium can develop V-shaped, W-shaped, Y-shaped, and U-shaped shapes in short-time culture. It is an aerobic bacterium whose cell arrangement is observed, and which becomes short rod-shaped or spherical when cultured for a long period of time.It is also recognized as belonging to the genus Arthrobacter due to its sugar usage characteristics. Manual of Determinative
Bacteriology) 7th edition (1957) and 8th edition
(1974), and among the seven species described in the genus Arthrobacter, those similar to Arthrobacter citreus,
Differences are recognized in the points listed in Table 4 below.

【table】

[Table] As shown above, this strain B-0780 is recognized as belonging to the genus Arthrobacter, but there is no other strain that closely matches the characteristics of this strain, so this strain is classified as Arthrobacter spp.・B-B-0780
(Arthrobacter sp.
FERM-P No.5663”). The novel L-arginine oxidase of the present invention may be one that has the above-mentioned substrate specificity and enzymatic action, and even if it shows some differences in molecular weight, Km value, isoelectric point, etc. Those having these properties and actions are included in the scope of the present invention, and the above-mentioned bacteria are examples of the bacteria used to obtain this novel L-arginine oxidase. In addition, the bacteria used in the production method of the present invention include:
The above-mentioned bacteria are just one example, and any bacteria belonging to the genus Arthrobacter that has the ability to produce L-arginine oxidase can be used in the present invention. Of course, microorganisms tend to mutate naturally or artificially, and even these mutant strains may
- It can be used in the present invention as long as it does not lose its ability to produce arginine oxidase. In carrying out the present invention, examples of L-arginine oxidase-producing bacteria belonging to the genus Arthrobacter are as follows. That is, L-arginine oxidase-producing bacteria belonging to the genus Arthrobacter are cultured by a conventional method for producing antibiotics, enzymes, and the like. The form of culture may be either liquid culture or solid culture, but from an industrial perspective it is advantageous to inoculate cells of L-arginine oxidase producing bacteria into a production medium and perform deep aeration agitation culture. As the nutrient source for the medium, those commonly used for culturing microorganisms are widely used. The nitrogen source may be any available nitrogen compound, such as corn steep liquor, soy flour, casein, peptone, enzyme extracts, various meat extracts, arginine, and various amino acids. The carbon source may be any assimilable carbon compound, such as molasses, glucose, starch hydrolyzate, glycerin, sucrose, and the like. In addition, various salts such as common salt, potassium chloride, magnesium sulfate, primary potassium phosphate, and secondary potassium phosphate, and antifoaming agents are used as necessary. The temperature of the medium can be changed as appropriate within the range that allows the bacteria to grow and produce L-arginine oxidase, but is particularly preferably 25 to 30°C. The culture time varies somewhat depending on the conditions, but is usually about 24 to 72 hours, and the culture may be terminated at an appropriate time, taking into account the time when L-arginine oxidase reaches its maximum titer. In the thus obtained culture of L-arginine oxidase-producing bacteria, most of L-arginine oxidase is contained and accumulated within the cells. In order to extract L-arginine oxidase from the culture obtained in this way, for example, the culture is first subjected to solid-liquid separation, and the obtained wet bacterial cells are separated using a solution such as a phosphate buffer. Crude L-
Obtain an arginine oxidase-containing solution. Next, this crude L-arginine oxidase-containing solution is further purified to obtain L-arginine oxidase using known isolation and purification means for proteins, enzymes, and the like. For example, a crude L-arginine oxidase-containing solution may be precipitated from an aqueous solution using a fractional precipitation method with an organic solvent such as acetone, methanol, ethanol, or isopropanol, or the L-arginine oxidase-containing solution may be recovered. may be carried out by ion exchange methods such as diethylaminoethyl cellulose, diethylaminoethyl dextran gel, triethylaminoethyl dextran gel, or adsorption chromatography using a gel filtration agent such as polyacrylamide gel. They may be combined as appropriate and purified until they show a single band by electrophoresis or the like, and then dried by freeze-drying or the like to obtain a purified powder of L-arginine oxidase. The novel L-arginine oxidase obtained in this way has L-arginine oxidase, as shown in its substrate specificity.
- Leucine aminopeptidase (hereinafter referred to as LAP) because it acts strongly on arginine and does not act on L-leucyl-L-arginine.
It is an extremely useful diagnostic enzyme for activity measurement. That is, L-leucyl-L, which is a synthetic substrate for LAP,
-L-arginine produced from a synthetic substrate by its LAP activity by acting on arginine,
This L-arginine oxidase is activated,
Next, the components of oxygen consumed or hydrogen peroxide and ammonia produced in this reaction system are
The activity of LAP is measured by quantifying using known electrical measuring means, such as oxygen electrodes, hydrogen peroxide electrodes, ammonia electrodes, other coloring means for hydrogen peroxide, and fluorescent means. At this time, L-arginine oxidase does not have any effect on its synthetic substrate, but acts only on L-arginine produced from the synthetic substrate based on LAP activity, so it is difficult to measure LAP activity very accurately. It is possible. Further, L-arginine oxidase is not limited to its usefulness as an enzyme for measuring LAP activity, but can also be used as an enzyme for analyzing L-arginine in food components, fermentation liquid components, etc., and for various other purposes. Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto in any way. Examples Polypeptone 1.0%, yeast extract powder 1.0%, glucose 1.0%, NaCl 0.5%, MgSO ₄ 0.05%,
K ₂ HPO ₄ 0.05%, Ehrlitsu meat extract 1.0%, milk casein 1.0%, arginine 1.0%, antifoaming agent 0.2
Medium consisting of % (autoclave sterilized, PH6.7)
Arthrobacter SB B-0780 strain was inoculated into 500 ml Erlenmeyer flasks (two bottles) containing 100 ml, and cultured with shaking at 30°C for 3 days to obtain a seed culture. Then 30 with 20 medium with the same composition
Two cultures of this kind were inoculated into a container jar and incubated at 30°C for 2 days at 200 rpm and aeration rate of 20/min.
The cells were cultured under the following conditions. After completion of the culture, the culture was centrifuged (5000 rpm, 10 minutes) to obtain wet bacterial cells, and the bacterial cells were washed twice with 10 mM phosphate buffer (PH6.0). Next, the wet bacterial cells were diluted with 5mM
After dispersing in 20mM phosphate buffer (PH6.0) containing EDTA, lysozyme was added so that the lysozyme concentration was 1 mg/ml. Thereafter, this was centrifuged (5000 rpm, 10 minutes) to obtain supernatant 2 (10000 U). Next, the supernatant was charged to a diethylaminoethyl cellulose column (6 x 30 cm) packed with 10 mM phosphate buffer (PH6.0), and the
After washing with 10mM phosphate buffer (PH6.0), 0-
Elution was performed using a linear concentration gradient method using 10 mM phosphate buffer (PH6.0) containing 0.5 M KCl, and the active fraction eluted with 10 mM phosphate buffer containing approximately 0.3 M KCl was collected. Ultrafiltration membrane XM
-50 (manufactured by Amicon). Next, this concentrated solution was passed through a Sephacryl S-200 column (3.6×
80cm), elute with 10mM phosphate buffer, collect the eluate between 500 and 600ml,
This was freeze-dried to obtain 958 mg (1380 U, 1.44 U/mg) of L-arginine oxidase powder.

[Brief explanation of the drawing]

Figure 1 shows the optimal pH for L-arginine oxidase.
The curve, Figure 2 shows the pH of L-arginine oxidase.
Stability curves: Figure 3 shows the thermostability curve of L-arginine oxidase, and Figure 4 shows the optimum temperature curve of L-arginine oxidase.

Claims (1)

[Claims] 1. At least the following substrate specificity, enzymatic action,
A novel L-arginine oxidase with molecular weight, Km value, isoelectric point, optimum PH and PH stability. Substrate specificity: Has substrate specificity for L-arginine. Enzyme action: It has an enzymatic action that strongly catalyzes the reaction shown by the following reaction formula []. Molecular weight: 140000±14000 Km value: 25×10 ^-3 M±2.5×10 ^-3 (for L-arginine) Isoelectric point: PH4.8±0.4 Optimal PH: Around 6.5 PH stable value: PH6.0 Nearby 2 L-arginine oxidase-producing bacteria belonging to the genus Arthrobacter are cultured, and L-arginine oxidase-producing bacteria belonging to the genus Arthrobacter are cultured, and L
- A method for producing a novel L-arginine oxidase having at least the following substrate specificity, enzymatic action, molecular weight, Km value, isoelectric point, optimum PH and PH stability, which comprises collecting arginine oxidase. Substrate specificity: Has substrate specificity for L-arginine. Enzyme action: It has an enzymatic action that strongly catalyzes the reaction shown by the following reaction formula []. Molecular weight: 140000±14000 Km value: 25×10 ^-3 M±2.5×10 ^-3 (for L-arginine) Isoelectric point: PH4.8±0.4 Optimal PH: Around 6.5 PH stable value: PH6.0 3. The method for producing L-arginine oxidase according to claim 2, wherein the L-arginine oxidase-producing bacteria belonging to the genus Arthrobacter is Arthrobacter sp. B-0780 strain.