JPH07123990A - Production of 2,5-dihydroxypyridine and synthetic enzyme for 2,5-dihydroxypyridine - Google Patents

Abstract

PURPOSE:To provide a method for mass-producing 2,5-dihydroxypyridine utilized as a raw material, etc., for agrochemicals in industrially utilizable high purity and obtain an enzyme for synthesizing the 2,5-dihydroxypyridine. CONSTITUTION:This method for producing 2,5-dihydroxypyridine is to convert 6-hydroxynicotinic acid into the 2,5-dihyroxypyridine using a microorganism which belongs to the genus Pseudomonas. This microorganism is Pseudomonas fluorescens and Pseudomonas fluorescens CSM-775 (FERM P-13924) deposited in the National Institute of Bioscience and Human-Technology, the Agency of the Science and Technology. Furthermore, this enzyme for synthesizing the 2,5-dihydroxypyridine is an enzyme produced by the Pseudomonas fluorescens CSM-775 (FERM P-13924).

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.

[0002]

2. Prior Art and Problems to be Solved by the Invention
5-Dihydroxypyridine is organically synthesized using 2-hydroxypyridine or 3-hydroxypyridine as a raw material. However, in this synthetic method, it is difficult to regioselectively hydroxylate, and various by-products are produced. Therefore, the yield is about 34% by weight when 2-hydroxypyridine is used as a raw material, and 3-hydroxypyridine is used. When used as a raw material, about 11% by weight [E. J. Behrman and
B. M. Pitt, Journal of the
American chemical Societ
y, 80, p. 3717-3718, 1958. reference〕
And extremely low. For this reason, conventionally, no industrially-available method for producing highly pure 2,5-dihydroxypyridine has been established.

On the other hand, 2,5-dihydroxypyridine is
Known as an intermediate in microbial metabolism of picolinic acid, nicotinic acid, and hydroxypyridine [(1)
O. P. SHUKLA et. al. Indian J
individual of Biochemistry &
Biophysics 10, p. 176-17819
73. (2) R. C. GUPTA et. al. Ind
ian Journal of Biochemisr
ty & Biophysics 15, p. 462-
464,1978. (3) C.I. HOUGHTON e
t. al. Biochem. J. 130, p. 879-
893,1972. reference〕. Since the intermediate 2,5-dihydroxypyridine is further metabolized and decomposed by microorganisms, no significant accumulation has been observed to date.

The present invention relates to a method capable of producing 2,5-dihydroxypyridine in industrially usable high purity and in a large amount, and an enzyme for synthesizing this 2,5-dihydroxypyridine. The purpose is to propose.

[0005]

DISCLOSURE OF THE INVENTION As a result of studies to achieve the above object, the present inventors have found that as an effective method for producing 2,5-dihydroxypyridine for industrial production, 2,5 -If a microorganism excellent in the production of dihydroxypyridine or an enzyme involved in it is used, 2,5-
The inventors have found that dihydroxypyridine can be produced with high efficiency and have completed the present invention.

[0006] Specifically, the present inventors first of all, from a large number of microorganisms, Pseudomonas fluorescens having the ability to produce 2,5-dihydroxypyridine from 6-hydroxynicotinic acid as a raw material.
Was found by separating from soil. Then, an enzyme capable of catalyzing the decarboxylation and hydroxylation of the carboxyl group of 6-hydroxynicotinic acid possessed by this microorganism to produce 2,5-dihydroxypyridine is treated with this 2,5-dihydroxypyridine. We have succeeded in preparing it with high purity and high efficiency so that it can be economically used for the production reaction of.

The method for producing 2,5-dihydroxypyridine of the present invention has been made on the basis of the results of these studies, and Pseudomonas fluoresc.
Using 6-hydroxynicotinic acid with ens
It is characterized by conversion into dihydroxypyridine.

In this case, the above-mentioned microorganism is Pseed deposited at the Institute of Biotechnology, Institute of Biotechnology, AIST.
omonas fluorescens CSM-77
It is also characterized in that it is 5 (FERMP-13924).

The synthase of 2,5-dihydroxypyridine of the present invention is the above-mentioned Pseudomonas f
luorescens CSM-775 (FERMP-
13924), which is an enzyme produced by 13924), that is, 6-hydroxynicotinic acid monooxygenase.

The microorganism used in the present invention is not particularly limited as long as it acts on 6-hydroxynicotinic acid to produce 2,5-dihydroxypyridine. Pseudomonas fluo deposited at the Institute of Biotechnology
rescens CSM-775 strain (FERMP-13
924).

For searching for a microorganism which produces 2,5-dihydroxypyridine, an ordinary method such as an enrichment culture method can be used. Specifically, the soil or test strain is
Enrichment culture is performed using a medium containing hydroxynicotinic acid.

Microorganisms producing 2,5-dihydroxypyridine can be analyzed by measuring the accumulated amount of 2,5-dihydroxypyridine in the culture medium by HPLC (high performance liquid chromatography) or TLC (thin layer chromatography). Selection can be made by using the amount as an index. The selected microorganism (that is, the 2,5-dihydroxypyridine-producing bacterium) can be further purified by using an agar medium or the like, if necessary.

Pseudomonas fluores
cens CSM-775 strain (FERMP-1392
4) is a strain isolated from the soil by an integrated culture method as a microorganism that accumulates 2,5-dihydroxypyridine in the culture solution by culturing in a medium containing 6-hydroxynicotinic acid. This strain has the following properties.

(A) Morphological properties: ・ Cell shape and size; bacilli, 1.0 to 3.0 μm ×
0.5-0.8 μm ・ Presence / absence of cell polymorphism; None ・ Presence / absence of motility; Yes ・ Growth state of flagella; Yes (polar flagella) ・ Presence or absence of spores ・ No

(B) Culture properties: ・ Meat broth agar plate culture; good growth, round, glossy light yellow brown ・ Meat broth agar slope culture; good growth, glossy, smooth and light brown color ・ Meat broth liquid culture ; Good growth, grows like a film on the liquid surface-Meat broth gelatin stab culture; Liquefied gelatin-Litmus milk; Alkaline, non-peptone

(C) Physiological properties: -Gram stainability; Negative-Reduction of nitrate; + -Denitrification reaction; + -MR test; -VP test; -Production of indole; -Production of hydrogen sulfide; Hydrolysis of starch-Use of citric acid-Media of Koser; -Media of Christensen-+-Use of inorganic nitrogen source-Nitrate; -Ammonium salt-+-Generation of fluorescent dyes- -Oxidase; + -catalase; + -range of growth; pH; 5.0-9.0 temperature; 4-37 ° C; attitude toward oxygen; aerobic; OF test; oxidative; decomposition of arginine;・ Anti-acidity; None ・ Use of various carbon sources; L-arabinose; -D-glucose; + D-mannose; + D-fructose; + D-galactose; + sucrose + Lactose; -trehalose; + D-mannite; + protocatechuic acid; + catechol; + gluconic acid;

With respect to the above-mentioned mycological properties, the Barjay's Manual of Systematic Bacteriology (Bargey's manual of Sys
The strain was found to be Pseudomona as a result of a search based on the textural biology.
s fluorescens.

As a medium for culturing these microorganisms,
Usually, any medium in which these microorganisms can grow is sufficient,
As the carbon source, sugars such as glucose and sucrose, organic acids such as acetic acid and succinic acid, and alcohols such as ethanol and glycerol are used. As the nitrogen source, inorganic salts such as sodium nitrate and ammonium sulfide, and organic compounds such as yeast extract, peptone, meat extract and urea can be used. In addition to these, inorganic salts, metal salts, vitamins and the like can be added as required.

In order to produce a large amount of 2,5-dihydroxypyridine synthase (ie, 6-hydroxynicotinic acid monooxygenase), 6-hydroxynicotinic acid, nicotinic acid or the like may be added. .
The amount of 6-hydroxynicotinic acid, nicotinic acid, etc. added is 0.01 to 10% by weight, more preferably 0.1 to 5% by weight, based on the medium (when adding two or more, the total It can be added so as to have a concentration of 1).

Culture temperature 20 to 40 ° C., more preferably 2
5 to 37 ° C., pH 5 to 9, more preferably 6 to 8,
It is desirable to culture aerobically. However, it is, of course, important to set the culture conditions depending on the microorganism used, the composition of the medium, etc. so that the production amount of 2,5-dihydroxypyridine is maximized.

The 2,5-dihydroxypyridine synthase of the present invention is purified as follows. First, a 2,5-dihydroxypyridine-producing bacterium is cultivated using the above-mentioned medium, and after the cells are grown, ultrasonic disruption, French press,
A crude enzyme solution is obtained by crushing the bacterial cells with dynamyl or by lysing the cell wall with a cell wall lysing enzyme such as rhizotium. From this crude enzyme solution, the synthesis enzyme of 2,5-dihydroxypyridine can be separated and purified by combining centrifugation, ammonium sulfate fractionation, ion exchange column chromatography, gel filtration and the like.

This enzyme acts on 6-hydroxynicotinic acid to decarboxylate the carboxyl group and at the same time acts as a catalyst for hydroxylation. Therefore, this enzyme is an enzyme that synthesizes 2,5-dihydroxypyridine, specifically 6-hydroxynicotinic acid monooxygenase, and requires NADH (nicotinamide adenine dinucleotide) and a molecular enzyme. .

The molecular weight of this enzyme is 42 KDa.
In addition to 6-hydroxynicotinic acid, this enzyme also acts on 2-hydroxynicotinic acid, hydroxybenzoic acid, etc. as shown in Table 1, although its relative activity is low. However, 2
-When acting on hydroxynicotinic acid or hydroxybenzoic acid, 2,5-dihydroxypyridine is not produced.

[0024]

[Table 1]

The relative activity is 25 ° C., pH 7.
When 6-hydroxypyridine and NADH are added and reacted in a buffer solution of 0, the enzyme activity that consumes 1 μmol of NADH in 1 minute is measured and calculated as 1 unit. The enzyme activity of the above 2,5-dihydroxypyridine synthase was 12.1 μmo per mg of protein.
It was 1 / min (25 ° C).

By reacting the 2,5-dihydroxypyridine synthase thus obtained with 6-hydroxynicotinic acid, highly pure 2,5-dihydroxypyridine can be obtained.

In the reaction at this time, 2,5-dihydroxypyridine synthase is suspended in a buffer solution having a pH of 6.0 to 9.0, preferably 7.0 to 8.0, and 6-hydroxynicotine is added thereto. Add 1 to 30 mM of acid, and add 10
To 50 ° C, more preferably 20 to 40 ° C for 10 minutes to
Do it for 24 hours. In addition, NA is used as a coenzyme in this reaction.
DH and FAD (flavin adenine dinucleotide) can also be added.

During the reaction, the reaction time and the like can be determined by analyzing the remaining amount of the starting material 6-hydroxynicotinic acid and the amount of 2,5-dihydroxypyridine produced by a conventional method such as HPLC analysis. When the remaining amount of -hydroxynicotinic acid decreases, 6-hydroxynicotinic acid may be further added to continue the reaction.

The 2,5-dihydroxypyridine can be recovered from the reaction solution by a conventional method. For example, by removing 2,5-dihydroxypyridine synthase by centrifugation, ultrafiltration, etc., concentrating the reaction solution, recrystallizing or solvent extraction with an organic solvent such as ethyl acetate, and evaporating to dryness, 2,5-dihydroxypyridine can be obtained.

[0030]

【Example】

Example 1 Pseudomonas fluo was added to the medium shown in Table 2.
inoculation with rescens CSM-775 strain at 30 ° C
The cells were cultured for 24 hours under aerobic conditions. After culturing, the bacterial cells were collected by centrifugation, and the pH was adjusted to 7.0 or 0.1.
The cells were suspended in M phosphate buffer, washed, and then resuspended in the same buffer to obtain a microbial cell concentrate. 0.35 ml of this microbial cell concentrate (hereinafter referred to as "mL", and the liter is referred to as "L"), the above buffer solution 1.45 mL, and 250 mM 6-
Mix with 0.2 mL of hydroxynicotinic acid aqueous solution,
The reaction was carried out at 25 ° C for 60 minutes. After the reaction, HP
When the amount of 2,5-dihydroxypyridine produced was analyzed by LC analysis, 20 mM of 2,5-dihydroxypyridine was detected.

[0031]

[Table 2]

Example 2 [Pseudomonas fluorescens]
Purification of 2,5-dihydroxypyridine synthase using CSM-775 strain (FERMP-13924)] In a medium containing 6-hydroxynicotinic acid shown in Table 3, Pse was used.
udomonas fluorescens CSM-
The 775 strain was cultured at 30 ° C. for 24 hours under aerobic conditions, and the cells were collected by centrifugation.

[0033]

[Table 3]

The collected strain was subjected to cell disruption for 10 minutes at 150 watts using an ultrasonic disruptor, followed by centrifugation at 100,000 g for 30 minutes. Ammonium sulfate fractionation is performed on the supernatant, and the ammonium sulfate concentration is 30 to 70% by weight.
Ion chromatograph (trade name "DEAE Sepharose CL manufactured by Pharmacia Co., Ltd.
-6B "was used. Furthermore, hydrophobic column chromatography (Pharmacia, trade name" Pheny1 ") was used.
Sepharose CL-4B ″) and purification by adsorption chromatography (Hydroxyapatite).

As a result, when the fractions having 2,5-dihydroxypyridine producing activity were collected and the purity of the enzyme was assayed by SDS electrophoresis, only one band was observed and it was found to be a single purified product. Was confirmed.

Example 3 When the enzyme (2,5-dihydroxypyridine synthase) purified in Example 2 was reacted with 6-hydroxynicotinic acid, 2,5-dihydroxypyridine produced was 1
It was 8.5 mM. The amounts of 2,5-dihydroxypyridine synthase, 6-hydroxynicotinic acid used, and the type and amount of coenzyme used at this time are shown in Table 4.
The reaction was carried out for 60 minutes at 25 ° C. in a total volume of 3 mL with 0.1 M phosphate buffer at pH 7.0.

[0037]

[Table 4]

[0038]

According to the production method of the present invention, highly pure 2,5-dihydroxypyridine can be produced,
Moreover, since this 2,5-dihydroxypyridine is not further metabolized, it can be accumulated at a high concentration, and 2,5-dihydroxypyridine can be obtained with high efficiency.

According to the enzyme of the present invention, 2,5-dihydroxypyridine can be synthesized with high purity and high efficiency.

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Claims (3)

[Claims] 1. A Pseudomonas fluore
Use 6-hydroxynicotinic acid with scens
A method for producing 2,5-dihydroxypyridine, which comprises converting to 5-dihydroxypyridine. 2. The microorganism is a Pseudomonas fluo deposited at the Institute of Biotechnology, Institute of Biotechnology, AIST.
rescens CSM-775 (FERMP-139
24), 2,5-
A method for producing dihydroxypyridine. 3. Pseudomonas fluore
scens CSM-775 (FERMP-1392
A synthase of 2,5-dihydroxypyridine, which is produced by 4).