JPH08107791A - Production of 2,5-dihydroxypyridine and microorganism for producing 2,5-dihydropyridine - Google Patents

Abstract

PURPOSE: To profitably obtain the subject compound useful as a raw material for agrochemicals, etc., at the ordinary temperature under the ordinary pressure by allowing a microorganism belonging to the genus Azotobacter having a 2,5-dihydroxypyridine-producing activity to act on 3-hydroxypyridine. CONSTITUTION: This method for producing 2,5-dihydroxypyridine comprises inoculating a microorganism [e.g. Azotobacter SP-HP-30 (FERM P-14555)] capable of growing on 3-hydroxypyridine and having a 2,5-dihydroxypyridine- producing activity on a medium, culturing the microorganism at 30 deg.C for 17hr under an aerobic condition, centrifugally collecting the cells, suspending collected cells in a 0.1M phosphoric acid buffer solution having a pH of 7.0, adding a 3-hydroxypyridine aqueous solution to the suspension, and subsequently subjecting the mixture to a reaction at 30 deg.C for 20min. Thus, the objective 2,5-dihydroxypyridine used as a raw material for agrschemicals is profitably obtained at the ordinary temperature under the ordinary pressure without danger.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing 2,5-dihydroxypyridine used as a raw material for agricultural chemicals and the like, and a microorganism producing the 2,5-dihydroxypyridine.

[0002]

2. Description of the Related Art Conventionally, in order to produce 2,5-dihydroxypyridine, 2
A chemical synthesis method is known in which -hydroxypyridine or 3-hydroxypyridine is reacted in the presence of peroxodisulfuric acid and sulfuric acid to obtain 2,5-dihydroxypyridine.

However, it is difficult to introduce a hydroxyl group in a position-specific manner by such a chemical synthesis method.
The yield of dihydroxypyridine is as low as 11 to 34% [E. J. Behrman and B.I. M. Pit
t, Journal of the American
chemical Society, vol. 80,
p. 3717 to 3718, 1958], industrially practical manufacturing methods have not been established.

Dihydroxypyridine, particularly 2,5-dihydroxypyridine, is a substance useful as a raw material for agricultural chemicals, and an industrial production method thereof has been earnestly desired.

In the conventional chemical synthesis method, as described above, the step of regioselectively introducing a hydroxyl group into hydroxypyridine is the most difficult.

On the other hand, 2,5-dihydroxypyridine is
It is known as an intermediate in microbial metabolism of picolinic acid, nicotinic acid, and hydroxypyridine, and it is known that a position-specific hydroxylation reaction occurs in the enzymatic reaction of a microorganism (OP SHUKLA et. ．
al. Indian Journal of Bioc
chemistry & Biophysics 10,
p. 176-178, 1973, R.I. C. GUPTA
et. al. Indian Journalof Bi
chemistry & Biophysics 1
5, p. 462-464, 1978, C.I. HOUGHT
ON et. al. Biochem. J. 130. p.
879-893, 1972).

However, since 2,5-dihydroxypyridine is further metabolized and decomposed by microorganisms, its accumulated amount is low, and a production method for industrial use has not been established.

[0008] Further, by using a microorganism, industrially effective
As a method for producing 5-dihydroxypyridine, the present inventors previously prepared 6-hydroxynicotinic acid as a raw material, and
A method for producing 2,5-dihydroxypyridine using a microorganism belonging to the genus Pseudomonas and a 2,5-dihydroxypyridine synthase produced by the microorganism has been proposed (Japanese Patent Application Nos. 5-302392 and 6-5990).
No. 3).

The present invention uses a microorganism different from these previously proposed microorganisms to produce 2,5-dihydroxypyridine with high efficiency, and a method which has not been known so far.
The purpose is to propose a 5-dihydroxypyridine-producing bacterium.

[0010]

[Means for Solving the Problems] In order to achieve the above object, the present inventors first searched for a microorganism having the ability to produce 2,5-dihydroxypyridine with high efficiency, and found that Azotobacter Microorganisms belonging to the genus, whose existence has not been known so far, are
It was found that it can grow on hydroxypyridine and has the ability to accumulate 2,5-dihydroxypyridine.

Next, the inventors of the present invention, using the above-mentioned microorganisms or other microorganisms belonging to the genus Azotobacter and having the above-mentioned characteristics, use 2,3-dihydroxypyridine as a raw material to enhance 2,5-dihydroxypyridine. We have obtained the knowledge that it can be manufactured with high efficiency.

The present invention was made based on the above findings, and is characterized by using a microorganism belonging to the genus Azotobacter and 3-hydroxypyridine.
The gist is a method for producing 5-dihydroxypyridine. In this production method, the above-mentioned microorganism can be produced by Azotobacterium sp.
The one deposited as HP-30 (FERM P-14555) is preferred.

The present invention is capable of growing on 3-hydroxypyridine and has an activity of producing 2,5-dihydroxypyridine.
obactor sp. HP-30 (FERM P-1
The gist of 2,5-dihydroxypyridine-producing bacteria deposited as 4555) is also included.

The bacterium of the present invention and the method for producing 2,5-dihydroxypyridine will be described in detail below. The bacterium of the present invention is
By introducing a hydroxyl group at the 6-position of 3-hydroxypyridine, 2,
It is a microorganism belonging to the genus Azotobacter that accumulates 5-dihydroxypyridine, and one example thereof is Azotobacterium sp. Which has been deposited as FERM P-14555 at the Institute of Biotechnology, Institute of Biotechnology, AIST. HP-3
0 shares can be mentioned.

The bacterium of the present invention was isolated from soil by the present inventors as a microorganism that accumulates 2,5-dihydroxypyridine when growing on 3-hydroxypyridine.

The microorganisms that can grow on such 3-hydroxypyridine and have the activity of producing 2,5-dihydroxypyridine can be obtained by a general screening method such as an integration culture method. Specifically, a microbial source such as soil or rainwater is cultivated in a medium containing 3-hydroxypyridine in a medium containing a nutrient source necessary for the growth of general microorganisms, and the grown microorganism is 2,5-dihydroxy. It can be carried out by determining whether or not it has an activity of producing pyridine. It can also be carried out by adding 3-hydroxypyridine in advance during the culturing, and measuring the decrease thereof or the amount of 2,5-dihydroxypyridine produced during the culturing.

An example of this screening method will be described below. A medium supplemented with a medium component necessary for bacterial growth and 3-hydroxypyridine is dispensed into a test tube, and after sterilization, a microbial source such as collected soil or rainwater is added, followed by shaking or static culture. Next, this culture solution is appropriately diluted and spread on an agar medium or the like to obtain colonies of grown bacteria. The obtained colonies are again cultivated using the same medium as above, and the culture solution is subjected to, for example, high performance liquid chromatography (hereinafter, HPLC), and the residual concentration of 3-hydroxypyridine, 2,5-dihydroxypyridine Is analyzed to select microorganisms capable of converting 3-hydroxypyridine to 2,5-dihydroxypyridine.

Incidentally, 3-hydroxypyridine, 2,5-
In the case of a bacterium having a strong activity of rapidly metabolizing both dihydroxypyridine, and when the amount of 2,5-dihydroxypyridine accumulated cannot be accurately measured by the above-mentioned analysis method, the bacterium after culturing is 2,5-dihydroxypyridine producing bacterium is selected by reacting with 3-hydroxypyridine and measuring 2,5-dihydroxypyridine producing activity.

As a medium component necessary for bacterial growth, general medium components can be used. In particular,
As a carbon source, one or two of sugars such as glucose and sucrose, organic acids such as acetic acid and succinic acid, and alcohols such as ethanol and glycerol.
More than one kind can be mixed and used. As nitrogen sources, inorganic salts such as sodium nitrate, sodium sulfate, ammonium sulfate, yeast extract, peptone, meat extract,
Organic compounds such as urea and glutamic acid can be added. In addition to these carbon source and nitrogen source, if necessary,
Inorganic salts, metals, vitamins, etc. can also be added.

Since 3-hydroxypyridine is generally highly toxic to microorganisms, its concentration cannot be increased so much that it is 20 to 30 mM to the medium.
It is suitable that the concentration is moderate.

As culture conditions, the pH of the medium is about 3 to
8, preferably about 5 to 7, and the culture temperature is 15 to 40.
C., preferably 20 to 37.degree. Further, it is desirable to appropriately perform stirring and shaking so that the substrate is sufficiently supplied.

The bacterium of the present invention (HP30 strain) obtained from the soil by the above screening operation has the following mycological characteristics.

(A) Morphology 1) Shape and size of cells E. coli, 3.0-3.5 × 1.5-2.0 μm 2) No cell polymorphism 3) No motility 4) Spores Presence / absence None 5) Gram stainability Negative 6) Anti-acidity None

(B) Growth state in each medium 1) Meat broth agar plate culture Good growth, orange colony, (shape) round shape, (protrusion) semi-lenticular shape, (peripheral) whole edge, (glossy) glossy 2 ) Meat juice agar slope culture Good growth, (surface) smooth, (color) orange, (glossy) glossy 3) Meat juice liquid culture Medium growth, no pellicle 4) Meat juice puncture culture Do not liquefy gelatin 5) No litmus milk peptone, precipitation or coagulation

(C) Physiological properties 1) Nitrate reduction Negative 2) Denitrification reaction Negative 3) MR test Negative 4) VP test Negative 5) Indole formation does not form 6) Hydrogen sulfide formation does not form 7) Starch Hydrolysis Negative 8) Utilization of citric acid Utilization of Koser's medium Utilization of Christensen's medium Utilization 9) Utilization of inorganic nitrogen source Utilization of nitrate Utilization of ammonium salt Utilization 10) Generation of pigment-11) Urease-12) Oxidase + 13) Catalase + 14) Range of growth pH 6.0-9.0 Temperature 18-38 ° C 15) Attitude toward oxygen Aerobic 16) OF test Negative

[0026]

(D) Others Nitrogen fixation Positive, reducing nitrate to nitrogen

The above-mentioned mycological properties are described in the Berjay's Manual of Systematic Bacteriology (BERGEY'S MANUAL OF System).
Based on the results of comparison with such as Bacterial Biology, the bacterium of the present invention shows that Azotobacter (Az
It is recognized as a bacterium belonging to the genus
bacteria sp. HP-30 (FERM P-14
555).

The bacterium of the present invention converts 3-hydroxypyridine into 2,5-dihydroxypyridine and regiospecifically and highly efficiently produces 2,5-dihydroxypyridine.

The 2,5-dihydroxypyridine-producing bacterium obtained as described above, or other Azotob
A method for producing 2,5-dihydroxypyridine using a microorganism belonging to the genus Acter and producing 2,5-dihydroxypyridine is performed as follows.

2,5-dihydroxypyridine producing bacterium,
Under the same culture conditions as in the above screening, 12
The cells are cultured for 48 hours, preferably 17-20 hours, and the cells are obtained by a centrifuge or the like. Next, a reaction solution is prepared by resuspending with a phosphate buffer or the like. The bacterial cell concentration in this reaction solution was 1 milliliter (hereinafter,
"ML" and liters are described as "L") 4 to 4 per
It is desirable that the amount is 50 mg, preferably 10 to 30 mg.

3-Hydroxypyridine is added to this reaction solution at a concentration of 10 to 100 mM, preferably 20 to 30 mM, and the temperature is 10 to 40 ° C., preferably 15
The reaction is carried out at ~ 35 ° C for 10 to 120 minutes. that time,
It is desirable to appropriately stir so that the bacterial cells and the raw materials are mixed well. In addition, air or oxygen gas can be blown in to efficiently supply oxygen required for the reaction.

Further, during the culture for preparing the reaction solution or during the reaction, the residual concentration of 3-hydroxypyridine as a raw material is measured, and the raw material can be appropriately added to carry out the reaction more efficiently. At this time, since 3-hydroxypyridine exhibits strong toxicity to microorganisms as described above, it is suitable to adjust the concentration of addition thereof within the above range for one addition.

The production examples of 2,5-dihydroxypyridine are shown below, but the present invention is not limited to these examples.

[0035]

【Example】

Example 1 Azotobacterium s was added to 50 mL of the medium shown in Table 1.
p. The HP-30 strain was inoculated and cultured at 30 ° C. for 17 hours under aerobic conditions. After culturing, the bacterial cells were collected by centrifugation, suspended in 0.1 M phosphate buffer of pH 7.0 and washed, and then resuspended in the same buffer to obtain a concentrated bacterial cell solution.

0.35 mL of this microbial cell concentrate, 1.45 mL of the above buffer, and 0.2 mL of 200 mM 3-hydroxypyridine aqueous solution were mixed to bring the total volume to 2 mL (3-hydroxypyridine concentration was 20 mM). The reaction was carried out at 30 ° C. for 20 minutes.

After completion of the reaction, 2,5-
When the production amount of dihydroxypyridine was analyzed, 3.
2 mM of 2,5-dihydroxypyridine was accumulated.

[0038]

[Table 1]

[0039]

[Table 2]

Example 2 In 1.8 L of the medium shown in Table 3, Azotobacterium was used.
sp. The HP-30 strain was cultured under aerobic conditions at 30 ° C. for 17 hours. After culturing, the bacterial cells were collected by centrifugation, suspended in 0.1 M phosphate buffer of pH 7.0 and washed, and then resuspended in the same buffer to obtain a concentrated bacterial cell solution.

15 mL of this microbial cell concentrate, 50 mL of the above buffer, and 5 mL of 300 mM 3-hydroxypyridine aqueous solution were mixed to make the total volume 70 mL (3-hydroxypyridine concentration 21 mM), and 60 at 30 ° C. A reaction of minutes was performed.

After completion of the reaction, HPLC analysis revealed 2,5-
When the production amount of dihydroxypyridine was analyzed, it was 1
Accumulation of 5.3 mM 2,5-dihydroxypyridine.

[0043]

[Table 3]

Example 3 The reaction was carried out in the same manner as in Example 1 except that the medium was cultured in 50 mL of the medium shown in Table 3 for 2 hours, and the residual amount of 3-hydroxypyridine was measured during the reaction, and when it reached 10 mM or less. In 3
The reaction was continued while adding 3-hydroxypyridine to 0 mM. As a result, 27 mM of 2,5-dihydroxypyridine was accumulated.

[0045]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to introduce a hydroxyl group in a position-specific manner by utilizing the substrate specificity of the enzymatic reaction shown by the microbial reaction, and produce 2,5-dihydroxypyridine in a high yield. Not only this, but also the reaction can be carried out under normal temperature and normal pressure, which is industrially advantageous without danger.

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Claims (3)

[Claims] 1. Azotobacte
r) A microorganism belonging to the genus and a method for producing 2,5-dihydroxypyridine, which comprises using 3-hydroxypyridine. 2. The microorganism is Azotobacterium sp. HP-30 (F
The method for producing 2,5-dihydroxypyridine according to claim 1, which is deposited as ERM P-14555). 3. A method capable of growing on 3-hydroxypyridine,
It has 2,5-dihydroxypyridine production activity, and has Azotobacter
sp. A 2,5-dihydroxypyridine producing bacterium deposited as HP-30 (FERM P-14555).