JPH0740951B2 - Method for producing hydroxide of nitrogen-containing heterocyclic compound by microorganism - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a hydroxide of a nitrogen-containing heterocyclic compound selected from nicotinic acid, pyrazinic acid and pyridinecarboxylic acid by utilizing a microorganism.

[0002]

BACKGROUND OF THE INVENTION Hydroxides of nitrogen-containing heterocyclic compounds such as 5-hydroxynicotinic acid, 5-hydroxypyrazine-2-carboxylic acid and 6-hydroxypyridine-3-sulfonic acid are It is known to be useful as a synthetic raw material or an intermediate for various pharmaceuticals, but the method for producing the hydroxides of these nitrogen-containing heterocyclic compounds is mainly carried out by the organic synthesis method, and the method utilizing microorganisms is not so common. unknown. As a slightly known example, a method of producing 6-hydroxynicotinic acid from nicotinic acid using a microorganism of the genus Achromobacter has been reported (Japanese Patent Publication No. 60-
196193, Japanese Examined Patent Publication 60-196194). In general, the organic synthesis method has a problem that the substitution reaction is not quantitative, or an undesirable by-product is produced along with the reaction. On the other hand, the method utilizing microorganisms overcomes the drawbacks of these organic synthesis methods and can be produced at a relatively low cost with high purity and high yield.

[0003]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to find an improved method for a hydroxide of a nitrogen-containing heterocyclic compound by a microorganism, and as a result, other than the microorganism of the genus Achromobacter. Also, Comamonas (Comam
It was discovered that microorganisms belonging to the genus onas also have the ability to produce 6-hydroxynicotinic acid from nicotinic acid, and further, these microorganisms contain not only nicotinic acid but also pyrazic acid and pyridine-3-sulfonic acid. The inventors have found that they have the ability to hydroxylate nitrogen heterocyclic compounds and have completed the present invention.

That is, the present invention relates to a microorganism belonging to the genus Comamonas and capable of hydroxylating a nitrogen-containing heterocyclic compound such as nicotinic acid, pyrazinic acid or pyridinesulfonic acid, and particularly to the bacterial cell or a treated product of the bacterial cell or Using the culture medium, nicotinic acid, pyrazic acid or pyridine-3-
Provided is a method for producing a hydroxide of a nitrogen-containing heterocyclic compound, which comprises treating a sulfonic acid to give a corresponding hydroxide.

The microorganisms used in the method of the present invention include:
A microorganism belonging to the genus Comamonas, which contains 6 parts of nicotinic acid, pyrazic acid or pyridine-3-sulfonic acid, respectively.
-Hydroxynicotinic acid, 5-hydroxypyrazine-2
-Carboxylic acid or 6-hydroxypyridine-3-sulfonic acid are all included, and specific examples include Comamonas acidoborans.
acidovorans) NA2. Comamonas acidoborans NA2 was isolated and identified by the present inventor from nature, and was submitted to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology on February 25, 1991, to the Institute of Microbiology and Microbiology 12038 (FER).
MP-12038). The mycological properties of the above Comamonas acidoborans NA2 are as follows.

(A) Morphology (cultured in broth agar medium at 30 ° C. for 24 hours) Cell shape and size: bacillus, 0.8 to 1.1 μm ×
2.5-4.1 μm Cell polymorphism: No Motility: Yes (has polar flagella) Spore: No Gram stain: Indeterminate Acidic: Negative

(B) Growth condition in each medium (30 ° C., 2
Culturing for 4 hours) Meat broth agar plate culture: Colonies are circular with a diameter of 1 mm or less, regular, pale yellow in color, and have a smooth and glossy surface. Broth agar slant culture: Medium growth with pale yellow color, smooth and shiny surface. Broth liquid culture: The culture broth is uniformly turbid, and bacterial precipitation occurs at the bottom.

(C) Physiological Properties Nitrate Reduction: Positive Denitrification Reaction: Negative Indole Formation: Negative Starch Hydrolysis: Negative Pigment Formation: Negative Urease: Negative Oxidase: Positive Catalase: Positive Growth Range: pH 6- 8. Temperature 20-41 ℃ 10 Attitude toward oxygen: Aerobic

11 OF test: O 12 Urea hydrolysis: negative 13 Testosterone degradation: negative 14 Testosterone utilization: negative 15 NO ₃ → NO ₂ : positive 16 NO ₃ → N ₂ : negative 17 Acetamide alkalization: positive 18 Allantoin alkalization: Negative 19 Tartaric acid alkalization: Positive

20 Generation of acid and gas from sugar: Generation of acid Generation of gas D-glucose + -D-fructose + -D-galactose + -D-mannitol +-

Based on the above mycological properties, Bergy's Manual of Systematic Bacteriolog
When classified by y), this microorganism was identified as a comanas acidovorans, which is an aerobic gram-negative bacillus, has polar flagella and is motile, and other physiological properties. .

The culture of the genus Comamonas used in the present invention can be carried out under the medium and culture conditions generally used for culturing microorganisms. That is, as the medium, an ordinary nutrient medium containing a carbon source, a nitrogen source, inorganic salts and the like can be used. Sugars as a carbon source, organic acids, or amino acids such as glutamic acid, polypeptone as a nitrogen source,
Yeast extract, soybean hydrolyzate, ammonium sulfate, etc. as trace metal components iron, magnesium, potassium,
A medium containing manganese or the like as appropriate can be used. Culturing is performed at pH 6-8, 20-41 ° C, preferably 2
It is carried out under aerobic conditions for 1 to 3 days at a temperature of 5 to 32 ° C.

To carry out the method of the present invention, first, nicotinic acid, pyrazic acid or pyridine-3-sulfonic acid is cultured in an inorganic or organic solvent which does not inhibit the reaction, preferably in an aqueous solvent, as obtained above. Liquor, cells collected from the culture solution or a treated product of the cells (for example, washed cells, dried cells, frozen cells, crushed cells, autolyzed cells, ultrasonic treatment of cells) Substance, cell extract, etc.) to produce 6-hydroxynicotinic acid, 5-hydroxypyrazine-2-carboxylic acid or 6-hydroxypyridine-3-sulfonic acid, respectively.

The starting concentration of nicotinic acid, pyrazinic acid or pyridine-3-sulfonic acid was 0.1% (w / w).
v) The above may be used as long as the reaction is not hindered.
The conditions for the treatment with the culture solution, the cells collected from the culture solution or the treated product of the cells may be any temperature as long as the enzyme does not denature, and usually 5 to 50 ° C., preferably 3
0 to 37 ° C., pH 4 to 10, preferably 5 to 8
Is.

When nicotinic acid, pyrazinic acid or pyridine-3-sulfonic acid is added to the culture solution, it is desirable to add it during the logarithmic growth phase of the bacterium. When using bacterial cells,
Cells in the logarithmic growth phase or stationary phase cultured by the above method are centrifuged, washed with physiological saline, etc., and then suspended in an appropriate buffer solution to give nicotinic acid, pyrazic acid or pyridine-3-sulfonic acid. May be added.

Then, the product 6-hydroxynicotinic acid, 5-hydroxypyrazine-2-carboxylic acid or 6-hydroxypyridine-3-sulfonic acid is recovered from the treated culture solution or reaction solution. As a recovery method, an ordinary method known to those skilled in the art can be used. That is, for example, after removing bacterial cells and the like from the reaction solution by centrifugation or membrane separation, 6-hydroxynicotinic acid, 5-hydroxypyrazine-2-, which is a target compound, is purified by a purification means such as column chromatography or crystallization. The carboxylic acid or 6-hydroxypyridine-3-sulfonic acid can be recovered.

Next, the present invention will be described in more detail based on examples, but the present invention is not limited to these. Example 1 Production of 6-hydroxynicotinic acid by Comamonas acidoborans NA2: (1) Culture of microorganism Comamonas acidoborans NA2 was shake-cultured at 28 ° C. for 36 hours using the following medium A.

[0018] Medium A ingredient content (in the medium 1L) nicotinic acid 3g malic acid 10g malt extract 5g Yeast extract _{5g MgSO 4 · 7H 2 O 0.5g} K 2 HPO 4 1g metal solution * 10 ml

(Note) *: The composition (in 1 L of the solution) is as follows. CaCl ₂ .2H ₂ O 400 mg H ₃ BO ₃ 500 mg CuSO ₄ .5H ₂ O 40 mg KI 100 mg FeSO ₄ .7H ₂ O 200 mg MnSO ₄ .7H ₂ O 400 mg ZnSO ₄ .7H ₂ O 400 mg H ₂ MoO ₄ .2H ₂ O 200 mg

(2) Production of 6-hydroxynicotinic acid: The above culture solution was centrifuged to collect the cells, which was washed with physiological saline and used for the reaction. That is, the bacterial cells obtained from 1 L of the culture solution were treated with 10 mM sodium acetate buffer (pH 5.
0) to 50 ml and add nicotinic acid (900
(mM) was added in 9 batches, and the reaction was carried out while shaking at 35 ° C. After 64 hours from the start of the reaction, nicotinic acid was completely consumed, and the amount of 6-hydroxynicotinic acid produced reached 865.2 mM (conversion rate: 96.1%, 120.4 g of 6-hydroxyl / L of reaction solution). Accumulation of nicotinic acid).

The produced 6-hydroxynicotinic acid was identified by separating it as a crystal and then conducting elemental analysis, IR, N.
It was performed by MR and mass spectrometry.

Example 2 Production of 5-hydroxypyrazine-2-carboxylic acid by Comamonas acidoborans NA2: (1) Culture of microorganisms Comamonas acidoborans NA2 was cultured by the same method as in Example 1 (1), and the following steps were carried out. Used for.

(2) Production of 5-hydroxypyrazine-2-carboxylic acid The above culture solution was centrifuged to collect the cells, and the cells were washed with physiological saline and used for the reaction. That is, the bacterial cells obtained from 1 L of the culture solution were treated with 10 mM sodium acetate buffer (pH 5.
0) to 50 ml and add pyrazinic acid (900
(mM) was added in 9 batches, and the reaction was carried out while shaking at 35 ° C. Pyrazic acid was completely consumed after 50.5 hours from the start of the reaction, and the amount of 5-hydroxypyrazine-2-carboxylic acid produced reached 782 mM (conversion rate:
86.9%, and 109.6 g of 5-hydroxypyrazine-2-carboxylic acid was accumulated per 1 L of the reaction solution).

The 5-hydroxypyrazine-2-carboxylic acid thus produced was identified by elemental analysis, IR, NMR and mass spectrometry after separating it as crystals.

Example 3 Production of 6-hydroxypyridine-3-sulfonic acid by Comamonas acidoborans NA2: (1) Cultivation of microorganism Comamonas acidoborans NA2 was cultured in the same manner as in Example 1 (1), and the following steps were carried out. Used for.

(2) Production of 6-hydroxypyridine-3-sulfonic acid The above culture solution was centrifuged to collect the cells, which was washed with physiological saline and used for the reaction. That is, the bacterial cells obtained from 1 L of the culture solution were treated with 10 mM sodium acetate buffer (pH 5.
0) to 50 ml, and pyridine-3-sulfonic acid (700 mM) was added thereto in 7 batches, and the reaction was carried out while shaking at 35 ° C. After 48 hours from the start of the reaction, the pyridine-3-sulfonic acid was completely consumed, and the amount of 6-hydroxypyridine-3-sulfonic acid produced was 66.
The amount reached 0.7 mM (conversion rate: 94.4%, 115.7 g of 6-hydroxypyridine-3-sulfonic acid was accumulated per 1 L of the reaction solution).

The produced 6-hydroxypyridine-3-sulfonic acid was identified by elemental analysis, IR, NMR and mass spectrometry after separating it as crystals.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Keigo Omura Inventor Keigo Omura 61 Izuminocho Yoshida Izuminocho, Sakyo-ku, Kyoto City, Kyoto Prefecture 201 Izumi Yoshida Heights 201 (56) References JP-A-60-196193 (JP, A)

Claims (6)

[Claims] 1. A nitrogen-containing complex characterized by treating nicotinic acid, pyrazinic acid or pyridinesulfonic acid with a microorganism belonging to the genus Comamonas and capable of hydroxylating nicotinic acid, pyrazinic acid or pyridinesulfonic acid. A method for producing a hydroxide of a ring compound. 2. The method according to claim 1, wherein the microorganism is used in the form of bacterial cells, a treated product of bacterial cells or a culture solution. 3. The method according to claim 1 or 2, wherein the microorganism is Comamonas acidoboranus NA2. 4. The method according to claim 1, 2 or 3, wherein 6-hydroxynicotinic acid is produced from nicotinic acid. 5. The method according to claim 1, 2 or 3 for producing 5-hydroxypyrazine-2-carboxylic acid from pyrazinic acid. 6. A method for producing 6-hydroxypyridine-3-sulfonic acid from pyridine-3-sulfonic acid, 1.
The method according to 2 or 3.