JPH08107790A - Production of 2,5-dihydroxypyridine and microorganism for producing 2,5-dihydroxypyridine - Google Patents

Abstract

PURPOSE: To provide a method for producing 2,5-dihydroxypyridine used as a raw material for agrochemicals, etc., and further to obtain a 2,5- dihydroxypyridine-producing microorganism. CONSTITUTION: The 2,5-dihydroxypyridine is produced from 6-hydroxynicotinic acid in the presence of at least one of microorganisms belonging to the genera Serratia, Alcaligenes, and Comamonas. The microorganisms are preferably Alcaligenes faecalis IAM 12586, Alcaligdnes faecalis IFO 14479, Alcaligenes faecalis CSM-6 (FERM P-14554), Serratia marcescens IFO 12648, and Comamonas acidovorans CSM-25 (FERM P-14556).

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 2,5-dihydroxypyridine used as a raw material for agricultural chemicals and the like, and a microorganism producing 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.

Hydroxypyridine, especially 2,5-dihydroxypyridine, is a useful substance as a raw material for agricultural chemicals and the like, and establishment of 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, the accumulated amount of 2,5-dihydroxypyridine as an intermediate in microbial metabolism known so far is low, and a practical method for producing 2,5-dihydroxypyridine 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 microorganism belonging to the genus Pseudomonas and a method for producing 2,5-dihydroxypyridine using the 2,5-dihydroxypyridine synthase produced by the microorganism have been proposed (Japanese Patent Application Nos. 5-302392 and 6-59903).
issue).

The present invention provides a method for producing 2,5-dihydroxypyridine using a microorganism different from those previously proposed, and a microorganism producing 2,5-dihydroxypyridine which has hitherto been unknown. With the goal.

[0010]

[Means for Solving the Problems] In order to achieve the above-mentioned object, the present inventors first of all, of a microorganism having the ability to efficiently accumulate 2,5-dihydroxypyridine from 6-hydroxynicotinic acid as a raw material. When I searched, 6
By culturing in a medium containing -hydroxynicotinic acid, it was possible to obtain a previously unknown microorganism that accumulates 2,5-dihydroxypyridine.

Next, the present inventors have succeeded in developing a microorganism capable of growing on 6-hydroxynicotinic acid and having an activity of producing 2,5-dihydroxypyridine, a genus Serratia,
It was possible to produce 2,5-dihydroxypyridine from 6-hydroxynicotinic acid using a microorganism belonging to the genus Alcaligenes and the genus Commonas.

The present invention is based on these facts,
The genus Serratia, the genus Alcaligenes, the Comamonas
The summary is a method for producing 2,5-dihydroxypyridine, which comprises using at least one selected from microorganisms belonging to the genus Monas) and 6-hydroxynicotinic acid. In this production method, the microorganism is A
lcaligenes faecalis IAM 1
2586, Alcaligenes faecalis
IFO 14479, Alcaligenes faeca deposited at the Institute of Biotechnology, Institute of Biotechnology, AIST
lis CSM-6 (FERM P-14554), S
erratia marcescens IFO 12
648, Commamonas acidovorans CS deposited at the Institute of Biotechnology, Institute of Biotechnology, AIST
It is preferably M-25 (FERM P-14556).

Further, the present invention is capable of growing on 6-hydroxynicotinic acid, has an activity of producing 2,5-dihydroxypyridine, and has been deposited with the Institute of Biotechnology, Institute of Industrial Science and Technology, Alcaligenes faecalis CS.
M-6 (FERM P-14554), the same Comma
NAS acidovorans CSM-25 (FE
RM P-14556) is also the subject.

The bacterium of the present invention and the method for producing 2,5-dihydroxypyridine will be described in detail below.

The microorganism used in the production of 2,5-dihydroxypyridine of the present invention is a microorganism which produces 2,5-dihydroxypyridine from 6-hydroxynicotinic acid, and is of the genus Serratia or the genus Alcaligenes. , Comamonas (Com
selected from the microorganisms belonging to the genus
Alcalige deposited at the Fermentation Research Institute
nes faecalis IFO 14479, Se
rratia marcescens IFO1264
8. A deposited at the Institute for Molecular and Cellular Biology, University of Tokyo
lcaligenes faecalis IAM 1
2586 is preferred. These microorganisms are microorganisms that have been searched by culturing in a medium containing 6-hydroxynicotinic acid and accumulating 2,5-dihydroxypyridine as an index.

It is also possible to separate it from soil or the like. In the present invention, for example, Alcaligenes fa was isolated from soil as a 2,5-dihydroxypyridine-producing bacterium and deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology.
ecalis CSM-6 (FERM P-1455
4), Commonas acidovoransC
SM-25 (FERM P-14556) is mentioned.

To search for a microorganism that produces 2,5-dihydroxypyridine contained in soil or the like, a conventional method such as an enrichment culture method can be used. Specifically, the microorganisms are cultured in a medium containing 6-hydroxynicotinic acid in a medium containing a nutrient solution necessary for the growth of general microorganisms, and the grown microorganisms produce 2,5-dihydroxypyridine. Can be searched by determining whether or not It is also possible to search by adding 6-hydroxynicotinic acid in advance during the culture and measuring the decrease in the culture or the amount of 2,5-dihydroxypyridine produced during the culture.

An example of this screening method will be described below. A medium containing 6-hydroxynicotinic acid and a medium component necessary for bacterial growth was dispensed into a test tube, and after sterilization,
Add microbial sources such as collected soil and rainwater, and perform 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 described above, and the culture solution is subjected to high-performance liquid chromatography (hereinafter, HPLC) and the like to determine the residual concentration of 6-hydroxynicotinic acid and 2,5-dihydroxy. The accumulated amount of pyridine is analyzed, and a microorganism having the ability to convert 6-hydroxynicotinic acid into 2,5-dihydroxypyridine is selected.

Incidentally, 6-hydroxynicotinic acid, 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 microbial cell after culturing is 2,5-dihydroxypyridine producing bacterium is selected by reacting with 6-hydroxynicotinic acid and measuring 2,5-dihydroxypyridine producing activity.

As a medium component required for bacterial growth, general components can be used. Specifically, as the carbon source, one kind or a mixture of two or more kinds of sugars such as glucose and sucrose, organic acids such as acetic acid and succinic acid, alcohols such as ethanol and glycerol can be 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 sources and nitrogen sources, inorganic salts, metals, vitamins and the like can be added as required.

As culture conditions, the pH of the medium is about 3 to
8, preferably about 5 to 7, and the culture temperature is 20 to 40.
C., preferably 25 to 37.degree. Further, it is desirable to appropriately perform stirring and shaking so that the substrate is sufficiently supplied.

2,5-dihydroxypyridine producing bacterium CSM-6 obtained from soil by the above-mentioned screening
The strain, CSM-25 strain, has the following mycological characteristics.

[0023]

(B) Growth state in each medium 1) Meat agar plate culture CSM6 strain: Growth level: good growth Color of colony: slightly grayish white, dull gloss Colony uplift: slightly raised colony Surroundings: All edges Surface of colony: Smooth CSM25 strain: Growth level: good growth Color of colony: pale cream color, glossy Surrounding of colony: wavy Surface of colony: smooth

2) Meat broth agar slant culture CSM6 strain: degree of growth: good growth Surface: smooth color: slightly grayish white with dull gloss CSM25 strain: degree of growth: good growth Surface: smooth color: white Brownish, shiny

3) Liquid culture of broth CSM6 strain: good growth, precipitation, no formation of pellicle CSM25 strain: good growth, precipitation, no formation of pellicle

4) Meat juice gelatin stab culture CSM6 strain: do not liquefy gelatin CSM25 strain: do not liquefy gelatin

5) Litmus milk CSM6 strain: No precipitation / coagulation, no alkalization CSM25 strain: No precipitation / coagulation, no alkalization

(C) Physiological properties CSM6 strain CSM25 strain 1) Reduction of nitrate- + 2) Denitrification reaction--3) MR test--4) VP test--5) Formation of indole--6) Hydrogen sulfide Weak -7) Starch hydrolysis-8) Utilization of citric acid Utilization of Koser medium Utilization Utilization of Christensen medium Utilization Utilization 9) Utilization of inorganic nitrogen source Utilization of nitrate Utilization Utilization of ammonium salt Utilization Utilization 10) Pigment formation--11) Urease- + 12) Oxidase + + 13) Catalase + + 14) Growth range pH 5.6 to 9.8 5.0 to 9.4 Temperature ° C 11 to 11 458-43 15) Attitude toward oxygen Aerobic and aerobic 16) OF test ---

17) Production of Acid and Gas from Sugar CSM6 Strain CSM25 Strain Substrate Acid Production Gas Production Acid Production Gas Production 1 L-arabinose -2-D-xylose -3-D-glucose ----- −4 D-mannose − − − − 5 D-fructose − − + − 6 D-galactose − − − − 7 Maltose − − − − 8 Sucrose − − − − 9 Lactose − − − − 10 Trehalose − − − − 11 D-Sorbit − − − − 12 D-Mannitol − − + − 13 Inosit − − + − 14 Glycerin − − + − 15 Starch − − − −

(D) Others CSM6 strain: m-, p-hydroxybenzoic acid does not grow, sodium acetate grows, TWEEN 80 does not hydrolyze CSM25 strain: acetamide, glycolate, L-histidine, m-, p -Grows on hydroxybenzoic acid

The above-mentioned mycological properties are described in the Berjay's Manual of Systematic Bacteriology (BERGEY'S MANUAL OF System).
magic Bacteriology)), The
From the results of comparison with Prokaryotes 2nd edition (1991), etc., CSM-6 strain is Alcalig.
enes faecalis and CSM-25 strain are Co
identified as mamonas acidovorans,
Each of these has been deposited at the Institute of Biotechnology, Industrial Technology Institute.

These bacteria of the present invention convert 6-hydroxynicotinic acid into 2,5-dihydroxypyridine and produce 2,5-dihydroxypyridine regiospecifically and highly efficiently.

The 2,5-dihydroxypyridine-producing bacterium thus obtained, or A deposited at the aforementioned Fermentation Research Institute or the Institute for Molecular Cell Biology, University of Tokyo
lcaligenes faecalis IAM 1
2586, Alcaligenes faecalis
IFO 14479, Serratia marce
scens IFO 12648, other Serratia (S
erratia genus, Alcali gene (Alcali)
gene) and a microorganism belonging to the genus Comamonas (hereinafter, these may be collectively referred to as "2,5-dihydroxypyridine-producing bacterium"), and a method for producing 2,5-dihydroxypyridine is as follows. Is done like.

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", liter is "L") 3 per
It is desirable that the amount be 50 mg, preferably 3 to 20 mg.

6-Hydroxynicotinic acid is added to this reaction solution at a concentration of 10 to 200 mM, preferably 10 to 100 mM, and the reaction is carried out at a temperature of 10 to 40 ° C., preferably 15 to 35 ° C. ~ 120 minutes. At 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 6-hydroxynicotinic acid as a raw material is measured, and the raw material is appropriately added, so that the reaction can be performed more efficiently. .

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

[0039]

【Example】

Example 1 In 50 mL of the medium shown in Table 1, Alcaligenes was added.
faecalis IAM 12586, Alcali
genesis faecalis IFO 14479,
Alcaligenes faecalis CSM-
6 (FERMP-14554), Serratia m
arcescens IFO 12648, Comam
onas acidovorans CSM-25 (F
ERMP-14556) strains were inoculated respectively at 30 ° C.
The cells were cultured for 18 hours under aerobic conditions.

After culturing, the bacterial cells were collected by centrifugation, suspended in 0.1 M phosphate buffer of pH 6.5 and washed, and then resuspended in the same buffer to obtain a concentrated bacterial cell solution. Obtained.

0.35 mL of this microbial cell concentrate (dry cell weight: 8 mg), 1.45 mL of the above buffer solution, and 200
0.2 mL of 6-hydroxynicotinic acid aqueous solution of mM was mixed to make the total volume 2 mL (6-hydroxynicotinic acid concentration was 20 mM), and the reaction was carried out at 30 ° C. for 20 minutes.

After completion of the reaction, HPLC analysis revealed 2,5-
When the production amount of dihydroxypyridine was analyzed, the amounts of 2,5-dihydroxypyridine shown in Table 3 were accumulated.

[0043]

[Table 1]

[0044]

[Table 2]

[0045]

[Table 3]

Example 2 In 50 mL of the medium shown in Table 4, Alcaligenes
faecalis CSM-6 strain, Commonas
As a result of carrying out the reaction in the same manner as in Example 1 except that the C. acidovorans CSM-25 strain was cultivated, respectively, the accumulated amount of 2,5-dihydroxypyridine was 12.5 mM (CSM-6 strain), 7 respectively. 0.1 mM (C
SM-25 strain).

[0047]

[Table 4]

Example 3 The residual amount of 6-hydroxynicotinic acid during the reaction was measured,
Reaction was performed in the same manner as in Example 2 except that the reaction was carried out for 6 hours while repeatedly adding 6-hydroxynicotinic acid so that the concentration of 6-hydroxynicotinic acid became 30 mM when the concentration became 10 mM or less. As a result,
The accumulated amount of 5-dihydroxypyridine is 22 m each
M (CSM-6 strain) and 15 mM (CSM-25 strain).

[0049]

As described above in detail, in the present invention, 6-
A large amount of 2,5-dihydroxypyridine can be accumulated by metabolism of a microorganism capable of causing a site-specific hydroxylation reaction with respect to hydroxynicotinic acid. Therefore, according to the present invention, practical production of 2,5-dihydroxypyridine can be performed.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12R 1:01) (C12N 1/20 C12R 1:05) (C12N 1/20 C12R 1:01) (72) Inventor Koji Hotta 1134-2 Gongendo, Satte City, Saitama Cosmo Research Institute's R & D Center

Claims (3)

[Claims] 1. At least one selected from microorganisms belonging to the genus Serratia, the genus Alcaligenes, the genus Comamonas, and 2,5-dihydroxy, which is characterized by using 6-hydroxynicotinic acid. A method for producing pyridine. 2. The microorganism is Alcaligenes fa
ecalis IAM 12586, Alcalige
nes faecalis IFO 14479, Alcal deposited at Institute of Biotechnology, Institute of Biotechnology, AIST
igenesfaecalis CSM-6 (FERM
P-14554), Serratia marces
cens IFO 12648, Commonas aci deposited at Institute of Biotechnology, Institute of Biotechnology, AIST
dovorans CSM-25 (FERM P-14
556), The method for producing 2,5-dihydroxypyridine according to claim 1. 3. Alca which can grow on 6-hydroxynicotinic acid and has 2,5-dihydroxypyridine producing activity, and has been deposited at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science and Technology.
ligenes faecalis CSM-6 (FE
RM P-14554) and the same Commonas ac.
idovorans CSM-25 (FERM P-1
4,556) which is a 2,5-dihydroxypyridine producing bacterium.