JPH04304894A - Production of hydroxide of picolinic acid or pyrazinic acid by microorganism - Google Patents

Abstract

PURPOSE:To produce a hydroxide of picolinic acid or pyrazinic acid in high purity and yield by using a microorganism. CONSTITUTION:Picolinic acid or pyrazinic acid is treated with a microorganism (its cultured liquid, cell or treated cell) belonging to genus Alcaligenes to obtain 6-hydroxypicolinic acid or 6-hydroxypyrazine-2-carboxylic acid.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing picolinic acid or pyrazine hydroxide using microorganisms.

0002

[Prior Art and Problems to be Solved by the Invention] Hydroxides of nitrogen-containing heterocyclic compounds such as 6-hydroxypicolinic acid and 6-hydroxypyrazine-2-carboxylic acid are useful as raw materials or intermediates for the production of various pharmaceuticals. However, these hydroxides are produced exclusively by organic synthesis methods, and no method using microorganisms is known. Organic synthesis methods generally have problems such as requiring special equipment and producing undesirable by-products during the synthesis reaction. By-products become impurities and must be removed from the reaction product after the reaction is completed, making the operation complicated. On the other hand, methods using microorganisms overcome the drawbacks of these organic synthesis methods, and can be produced at relatively low cost with high purity and high yield.

0003

[Means for Solving the Problems] The present inventors have overcome the drawbacks of the above-mentioned organic synthesis methods, and have produced nitrogen-containing complexes such as 6-hydroxypicolinic acid and 6-hydroxypyrazine-2-carboxylic acid using microorganisms. In order to obtain hydroxides of ring compounds with high purity and high yield, we searched for various microorganisms and found that microorganisms belonging to the genus Alcaligenes have the ability to produce the above hydroxides from picolinic acid or pyrazine acid. This discovery led to the completion of the present invention.

That is, the present invention relates to the treatment of picolinic acid or pyrazine acid using a microorganism belonging to the genus Alcaligenes that is capable of hydroxylating picolinic acid or pyrazine acid, particularly its culture solution, bacterial cells, or a treated product of the bacterial cells. Provided is a method for producing hydroxides of picolinic acid and pyrazine acid, characterized in that the method leads to the corresponding hydroxides.

[0005] Microorganisms used in the method of the present invention include:
Any microorganism belonging to the genus Alcaligenes that converts picolinic acid or pyrazine acid into 6-hydroxypicolinic acid or 6-hydroxypyrazine-2-carboxylic acid may be used. A specific example is Alcaligenes faecalis (Alca
ligenes faecalis) PA6. Alcaligenes faecalis PA6 was isolated and identified from nature by the present inventor, and was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology on February 25, 1991, as part of the Microbiology Research Institute No. 12037 (FERM P -12037). The mycological properties of this microorganism are as follows.

(a) Morphology (cultivated on broth agar medium at 30°C for 24 hours) ■Cell shape and size: Bacillus, 0.5-1.2 μm x 0.5-
2.6μm ■Cell pleomorphism: No ■Motility: Yes (with unisexual flagellum) ■Spores: No ■Gram stainability: Negative ■Acid-fastness: Negative

(b) Growth status in each culture medium (30°C, 24-hour culture) ■ Broth agar plate culture: Colonies are circular with a diameter of 0.5 mm or less, regular, pale yellowish-white in color, and the surface is glossy. It's smooth. ■Juice agar slant culture: Medium growth, pale yellow-white color, smooth and glossy surface. ■ Broth liquid culture: The culture solution becomes uniformly cloudy and bacteria precipitates at the bottom. ■Meat juice gelatin puncture culture: Good growth in the upper layer and growth along the puncture site. ■Litmus milk: Produces alkali.

(c) Physiological properties ■ Nitrate reduction: negative ■ Denitrification reaction: negative ■ MR test: negative ■ VP test: negative ■ Indole formation: negative ■ Hydrogen sulfide formation: negative ■ Starch hydrolysis: Negative ■ Utilization of citric acid: Koser's medium; Positive Chr
istensen's medium; positive

■ Utilization of inorganic nitrogen sources: Nitrate; Positive Ammonium salt; Positive 10 Pigment production: Negative 11 Urease: Negative 12 Oxidase: Positive 13 Catalase: Positive 14 Growth range: pH 6-8, temperature 20-37°C 15
Attitude towards oxygen: aerobic

0010 16O-F test: negative Generation of acid and gas from 17 sugars:

Based on the above mycological properties, Bergy's Manual of Sy.
According to Stematic Bacteriology, this microorganism is an aerobic Gram-negative bacillus, positive for catalase, positive for oxidase, negative for the OF test, negative for acid production from glucose, and has a unisexual flagellum and is motile. Alcaligenes faecalis (Alcaligenes faecalis)
It was identified as ``Igenes faecalis''.

[0012] The above-mentioned microorganisms used in the present invention can be cultured using a medium and culture conditions generally used for culturing microorganisms. That is, as a medium, a normal nutrient medium containing a carbon source, a nitrogen source, inorganic salts, etc. can be used. Carbon sources include sugars such as fructose, glucose, lactose, starch, dextrin, and maltose, or amino acids such as glutamic acid. Nitrogen sources include polypeptone, yeast extract, soy flour hydrolyzate, and ammonium sulfate. Trace metal components include iron, magnesium, A medium containing potassium, manganese, etc. as appropriate can be used. Culture at pH 6-8, 20-37°C,
It is preferably carried out under aerobic conditions at a temperature of 25-32°C for 1-3 days.

To carry out the method of the present invention, first, picolinic acid or pyrazine acid is collected from the culture solution obtained above in an inorganic or organic solvent that does not inhibit the reaction, preferably an aqueous solvent. bacterial cells or processed products of the bacterial cells (for example, washed bacterial cells, dried bacterial cells, frozen bacterial cells, ground bacterial cells,
6-hydroxypicolinic acid or 6-hydroxypyrazine-2-carboxylic acid is produced by treatment with a bacterial cell autolysate, a bacterial cell sonication product, a bacterial cell extract, etc.), respectively.

The concentration of the starting material, picolinic acid or pyrazine acid, may be 0.1% (w/v) or more as long as it does not inhibit the reaction. The conditions for treatment with a culture solution, microbial cells collected from the culture solution, or processed products of the microbial cells are as long as the temperature does not denature the enzyme, and is usually 5 to 50°C, preferably 30°C. ~37°C, p
H is 5-9, preferably 7-8.

When picolinic acid or pyrazine acid is added to the culture solution, it is desirable to add it during the logarithmic growth phase of the bacteria. When using bacterial cells, the bacterial cells in logarithmic growth phase or stationary phase cultured by the above method are centrifuged, washed with physiological saline, etc., and then added to 10 mM potassium phosphate buffer (pH 7.
What is necessary is to suspend the bacterial cells in 5) and add picolinic acid or pyrazine acid.

Next, the product 6-hydroxypicolinic acid or 6-hydroxypyrazine-2-carboxylic acid is recovered from the treated culture solution or reaction solution. As a recovery method, conventional methods known to those skilled in the art can be used. That is, for example, after removing bacterial cells from the reaction solution by centrifugation, membrane separation, etc., the target compound 6-hydroxypicolinic acid or 6-hydroxypyrazine-2- is purified by purification means such as column chromatography or crystallization. Carboxylic acid can be recovered.

Next, the present invention will be explained in more detail based on Examples, but the present invention is not limited thereto. Example 1 Production of 6-hydroxypicolinic acid using Alcaligenes faecalis PA6: (1) Cultivation of microorganisms Using the following medium A, Alcaligenes faecalis PA
6 was cultured with shaking at 28°C for 36 hours.

[0018]

(Note) *: The composition (in 1 L of solution) is as follows. CaCl2・2H2O 400mgH3
BO3 500m
gCuSO4・5H2O 40mg
KI 1
00mgFeSO4・7H2O 200
mgMnSO4・7H2O 400mg
ZnSO4・7H2O 400mgH2
MoO4・2H2O 200mg

002
(2) Production of 6-hydroxypicolinic acid: The above culture solution was centrifuged to collect bacteria, washed with physiological saline, and used for reaction. That is, bacterial cells obtained from 1 L of culture solution were suspended in 10 mM potassium phosphate buffer (pH 7.5) to make 50 ml, picolinic acid (900 mM) was added in 9 portions, and the mixture was shaken at 35°C. I reacted slowly. 60 hours after the start of the reaction, picolinic acid was completely consumed, and the amount of 6-hydroxypicolinic acid produced was 8
It reached 32.3mM (conversion rate: 92.5%, reaction solution 1
115.8 g of 6-hydroxypicolinic acid accumulated per L).

The produced 6-hydroxypicolinic acid was identified by elemental analysis, IR, N
This was done by MR and mass spectrometry.

Example 2 Production of 6-hydroxypyrazine-2-carboxylic acid using Alcaligenes faecalis PA6: (1) Cultivation of microorganisms Alcaligenes faecalis PA6 was grown in Example 1 (1)
The cells were cultured in the same manner as described above and used in the following steps.

(2) Production of 6-hydroxypyrazine-2-carboxylic acid The above culture solution was centrifuged to collect bacteria, washed with physiological saline, and used for reaction. That is, bacterial cells obtained from 1 L of culture solution were added to 10 mM potassium phosphate buffer (pH 7).
．． 5) to make a total volume of 50 ml, and add pyrazine acid (7
00mM) was added in 7 portions, and the reaction was carried out at 35°C with shaking. 48 hours after the start of the reaction, pyrazine acid was completely consumed, and the amount of 6-hydroxypyrazine-2-carboxylic acid produced reached 631.8mM (conversion rate: 90.3%, 88.5g per 1L of reaction solution). 6-
(accumulation of hydroxypyrazine-2-carboxylic acid).

The produced 6-hydroxypyrazine-2-carboxylic acid was identified by elemental analysis, IR, NMR and mass spectrometry after separating it as crystals.

Claims (5)

[Claims] 1. A method comprising treating picolinic acid or pyrazine acid using a microorganism that belongs to the genus Alcaligenes and is capable of hydroxylating picolinic acid or pyrazine acid.
A method for producing picolinic acid or pyrazinic acid hydroxide. 2. The method according to claim 1, wherein the microorganism is used in the form of bacterial cells, a processed product thereof, or a culture solution. 3. The method according to claim 1 or 2, wherein the microorganism is Alcaligenes faecalis PA6. 4. The method according to claim 1, 2 or 3, wherein 6-hydroxypicolinic acid is produced from picolinic acid. 5. The method according to claim 1, wherein 6-hydroxypyrazine-2-carboxylic acid is produced from pyrazine acid.