JP2001046093A - Production of polyhydric alcohol-aromatic carboxylic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply produce the subject compound useful as an intermediate, etc., for medicines and agrochemicals by bringing a biological cell, a biological tissue, etc., capable of catalyzing a reaction for carboxylating an aromatic polyhydric alcohol into contact with the above polyhydric alcohol in the presence of carbonate ions, etc. SOLUTION: An aromatic polyhydric alcohol represented by formula I ((n) is 2 or 3) (e.g. resorcinol) is brought into contact with a tissue or a cell of an animal or a plant, a microorganism or a treated material of the tissue, cell or microorganism having the ability to provide a catalyst for a reaction in which the above compound is reacted with a carbonyl compound represented by the formula (CO2)m ((m) is 1 or 2) to produce a polyhydric alcohol-aromatic carboxylic acid represented by formula II (e.g. γ-resorcinolcarboxylic acid) in the presence of carbonate ions and/or CO2 in an aqueous solvent to produce the polyhydric alcohol-aromatic carboxylic acid represented by formula II in the reactional liquid. The resultant polyhydric alcohol- aromatic carboxylic acid is then separated from the reactional system to thereby simply and selectively produce the objective compound useful as an intermediate for medicines and agrochemicals, a raw material, etc. for liquid crystals, heat-sensitive papers, lithographic plates, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an aromatic polyhydric alcohol represented by the general formula (1) (formula 3) (hereinafter simply referred to as an aromatic polyhydric alcohol) utilizing the ability of organisms to fix carbon dioxide. To a polyhydric alcohol aromatic carboxylic acid represented by the general formula (2) (Chemical Formula 3) (hereinafter sometimes simply referred to as a polyhydric alcohol aromatic carboxylic acid). It relates to a method of manufacturing.

[0002]

Embedded image (Wherein, n represents 2 or 3, and m represents 1 or 2, respectively)

[0003] Polyhydric alcohol aromatic carboxylic acids are used as raw materials for intermediates for medical and agricultural chemicals, liquid crystals, thermal paper, lithographic plates and the like, and are industrially useful compounds.

[0004]

2. Description of the Related Art Hitherto, polyhydric alcohol aromatic carboxylic acids have been industrially produced mainly from aromatic polyhydric alcohols by a carbonation reaction by the Kolbe-Schmidt method. That is, the polyhydric alcohol aromatic carboxylic acid is produced by reacting a metal salt of an aromatic polyhydric alcohol with carbon dioxide at high temperature and high pressure. However, this manufacturing method involves danger because it is performed under high temperature and high pressure conditions,
In addition, since the polyhydric alcohol aromatic carboxylic acid is produced as a mixture of compounds having carboxylic acids added at various positions, a separate and complicated separation and purification step is required to obtain the target compound from the mixture. Various methods for selectively producing a desired polyhydric alcohol aromatic carboxylic acid by chemical synthesis have been studied, but have not been put to practical use.

[0005] To overcome the above disadvantages of the chemical synthesis method,
Multivalent alcohols of interest selectively under relatively mild conditions
In order to obtain an aromatic carboxylic acid, a method of producing a polyhydric alcohol aromatic carboxylic acid from an aromatic polyhydric alcohol utilizing the ability of a living body is considered. There is no known method. A method of producing phenol carboxylic acid by adding carbon dioxide to a phenolic compound which is a monohydric alcohol using a slight amount of microorganism is known (He, Z. and Wiegel, J .: J. Bacteriol). .,
vol.178, pp.3539-3548, (1996)). However, this method has a low reaction yield and is not suitable for industrial production of phenol carboxylic acid.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to utilize a biological function to easily and selectively convert an aromatic polyvalent alcohol into a desired polyvalent alcohol at ordinary temperature and pressure. To provide a method for producing an aromatic carboxylic acid.

[0007] Accordingly, an object of the present invention is to provide a tissue or cell, a microorganism, or a treated product of an animal or plant having the ability to produce a polyhydric alcohol aromatic carboxylic acid from an aromatic polyhydric alcohol. To provide a method for producing a polyhydric alcohol aromatic carboxylic acid from an aromatic polyhydric alcohol using the method.

[0008]

Means for Solving the Problems The present inventors have made intensive studies in order to solve the above-mentioned problems, and as a result, from the soil, belonged to the genus Propionibacterium and produced γ-resorcinic acid (2,6-dihydroxybenzoic acid). Bacteria that degrade to resorcin were isolated. When this bacterium is brought into contact with resorcin, which is an aromatic polyhydric alcohol in the presence of carbonate ions and / or carbon dioxide, γ-resorcinic acid, which is a polyhydric alcohol aromatic carboxylic acid, is selectively produced. I found that I got it. In addition, 2,3-dihydroxybenzoic acid was successfully produced from catechol using the same microorganism. The present invention has been made based on the above findings.

That is, the present invention is as follows. [1] Animal or plant tissues or cells, microorganisms, or microorganisms having the ability to catalyze the reaction of the aromatic polyhydric alcohol represented by the general formula (1) with the reaction represented by the reaction formula (1) (Formula 4) The treated tissue, cell or microorganism is brought into contact with an aqueous solvent in the presence of carbonate ions and / or CO ₂ , and a polyhydric alcohol aromatic carboxylic acid represented by the general formula (2) is added to the reaction mixture. And then separating the polyhydric alcohol aromatic carboxylic acid represented by the general formula (2) from the reaction system.

[0010]

Embedded image (Wherein, n represents 2 or 3 and m represents 1 or 2, respectively) [2] Animal or plant tissue or a plant having the ability to catalyze the reaction represented by the above reaction formula (1) (Formula 4) Cells, microorganisms, or processed products of the tissues, cells, or microorganisms. [3] An enzyme that catalyzes the reaction represented by the reaction formula (1) derived from an animal, a plant, or a microorganism having the ability to catalyze the reaction represented by the above reaction formula (1) (Formula 4).

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
In the present invention, those having the ability to catalyze the reaction represented by the reaction formula (1) include animal or plant tissues or cells having the ability to catalyze the reaction represented by the reaction formula (1). , Microorganisms, or processed products of the tissues, cells, or microorganisms.

Here, examples of animals include mammals such as humans, mice and rats, lepidopteran insects and the like, and examples of plants include solanaceous plants such as tobacco, gramineous plants such as rice, and Arabidopsis thaliana and the like. A cruciferous plant is exemplified.

The animal or plant tissue or cell is a tissue or cell directly separated from the organism.
Alternatively, it refers to a tissue of such an organism, a cultured tissue obtained by culturing cells, callus cells, cultured cells, and the like. Culture of animal or plant cells is known per se, for example, solid culture and liquid culture, and is exemplified by aromatic polyhydric alcohols such as resorcin, or polyvalent alcohols such as α-resorcinic acid and γ-resorcinic acid. It is effective to add alcohol aromatic carboxylic acids (benzoic acid derivatives) to the medium in which they are used, since the catalytic ability of the cells may be enhanced. The amount of the compound to be added is, for example, α-resorsinic acid or γ-resorsinic acid in the range of 1 to 100 mM, preferably 1 to 100 mM in the liquid medium.
It is preferable to add so as to be 0 to 50 mM. In addition, a recombinant animal or plant tissue or cell into which a gene encoding an enzyme involved in the reaction represented by the reaction formula (1) is introduced using a genetic engineering technique can also be used in the production method of the present invention. .

In the present invention, the microorganism having the ability to catalyze the reaction represented by the reaction formula (1) is a microorganism having the ability to catalyze the reaction represented by the reaction formula (1). Is exemplified. It is advantageous to use bacteria, particularly facultative anaerobic bacteria, in view of the fact that they have a high growth rate and a high growth rate even in the presence of carbon dioxide, and have high reactivity. Propionibacteria can be used as such facultative anaerobic bacteria.

The bacteria used in the present invention include Propionibacterium MT-10883 ( Propionibacterium) isolated from soil by the present inventors.
freudenreichii MT-10883).
The mycological properties of this strain are as follows.

[0016]

The inventors of the present invention with respect to the taxonomic properties of the strain
According to the detailed examination of the above, in addition to the above points,
When the CFA was measured, the branched antennae type
Of CFA were detected. Branched antennae-type CFA
Is normalCorynebacteriumNot found in. Plant pathogenicity
ofCorynebacteriumOnly known to exist
You. Plant pathogenicCorynebacteriumIndicates nitrate reduction
From where notCorynebacteriumThe possibility of being denied
Was. The branched anteiso-type CFA of the bacterium is C ₁₄But
Due to its occupancy, the bacteriumCorynebacteriumThen
WithoutPropionibacteriumIt was concluded thatPropionib
acteriumAs a result of a search for the properties ofPr
opionibacterium freudenreichiiAnd decidePropioniba
cterium freudenreichii MT-10883
Was.

This strain was submitted to the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, 1-3-1 Higashi, Tsukuba, Ibaraki Prefecture on June 8, 1999 under the accession number: FERM P-
No. 17415.

As the microorganisms used in the present invention, natural microorganisms may be subjected to a mutation treatment, and those having an increased productivity of polyvalent alcohol aromatic carboxylic acid may be selected and used. Such mutant microorganisms include, for example, mutant strains having improved resistance to aromatic polyhydric alcohols and polyhydric alcohol aromatic carboxylic acids, and the ability to degrade polyhydric alcohol aromatic carboxylic acids. And mutants having high productivity of enzymes involved in polyvalent alcohol aromatic carboxylic acids. In addition, it has the ability to produce a recombinant microorganism into which a gene encoding an enzyme involved in the reaction represented by the reaction formula (1) has been introduced using genetic engineering techniques, and to produce the enzyme constitutively or inducibly. A microorganism, an extract of the microorganism, or an isolated enzyme can also be used in the production method of the present invention.

The method for culturing microorganisms used in the present invention will be described below. The medium for growing the microorganisms is not particularly limited as long as it is a medium such as a solid medium or a liquid medium in which these microorganisms can grow, and specific examples include "Normal broth medium (manufactured by Eiken)" and the like. . As the carbon source added to the medium, any carbon compound that can assimilate and grow cells can be used. Examples thereof include sugars such as glucose and sucrose, and polyvalent alcohols such as glycerin. As a nitrogen source added to the medium, for example, ammonium sulfate, ammonium chloride,
Inorganic nitrogen sources such as ammonium nitrate and organic nitrogen sources such as yeast extract, peptone and meat extract can be used.
In addition to these, if necessary, inorganic salts, metal salts, vitamins and the like can be added to the medium.

The cultivation of the microorganism is carried out at a temperature of 20 to 40 ° C., preferably 25 to 35 ° C.
The treatment may be performed at 0.0, preferably at pH 7.0 to 9.0.
The cultivation can be performed either under anaerobic or aerobic conditions according to the characteristics of the microorganism to be cultured, but in the case of aerobic microorganisms, shaking culture under aerobic is preferable because the growth rate is high, It can be carried out under normal pressure. The culture period is several hours to 10 days, preferably 1 to 3 days.

The above culture conditions such as temperature, pH, aeration, time and the like can be appropriately changed according to the microorganism used, the composition of the medium and the like, but the production amount of the polyhydric alcohol aromatic carboxylic acid as the final product is determined. Is of course important to set the maximum value. In addition, in order to produce a large amount of an enzyme that converts aromatic polyhydric alcohols into polyhydric alcohol aromatic carboxylic acids, it is effective to add a benzoic acid derivative to a medium for growing cells. is there. As an example α
-Resorcinic acid or γ-resorcinic acid. The addition amount is 1 to 100 mM based on the culture medium,
It is preferable to add so that the concentration is preferably 10 to 50 mM. Microbial cells can be collected from a culture obtained by culturing, for example, in the case of culturing in a liquid medium, the culture solution may be centrifuged.

In the production method of the present invention, the above-mentioned tissues, cells or microorganisms obtained as described above may be used directly, but if necessary, processed products thereof can be used.
Here, the processed material of a tissue or a cell refers to the tissue,
Cell or microorganism crushed tissue, crushed cell, crushed cell, cell culture solution, cell culture solution, organic solvent-treated tissue, organic solvent-treated cell, organic solvent-treated cell, crude enzyme or purified enzyme, immobilized cell, fixed Examples thereof include those obtained by suspending a bacterium, a tissue cell, or a microorganism in an aqueous solvent containing an aromatic polyhydric alcohol. These tissues, cells or microorganisms and their processed products can be used for the reaction immediately after the production, or stored after the production, and the required amount can be used as needed.

The reaction method will be described below. Aromatic polyhydric alcohol used as a raw material in the production method of the present invention
Is a compound represented by the general formula (1) (Formula 4). Specifically, divalent aromatic polyhydric alcohols such as resorcin, catechol and hydroquinone, and trivalent aromatic polyhydric alcohols such as pyrogallol, phloroglucinol and 1,2,4-benzenetriol are used. is there. The aromatic polyhydric alcohol used in the reaction is added to the reaction solution at a concentration of 1 to 1000 mM, preferably 10 to 300 mM. The reaction is carried out by converting animal or plant tissues, cells, microorganisms or a treated product of the tissues, cells or microorganisms and an aromatic polyhydric alcohol into an aqueous solvent in the presence of carbonate ions and / or carbon dioxide (CO2).
₂ ) The polyhydric alcohol aromatic carboxylic acid is allowed to accumulate in the reaction mixture in the presence of ₂ ).

The method for producing a polyhydric alcohol aromatic carboxylic acid from an aromatic polyhydric alcohol can be a batch type or a continuous type using a bioreactor. Examples of the aqueous solvent used for the reaction include a buffer such as a carbonate buffer, preferably a potassium bicarbonate / potassium carbonate buffer having a concentration of 50 to 2000 mM. The amount of animal or plant tissue, cells, or microorganisms or the treated product of the tissue, cells or microorganisms added to the reaction solution is 0.1 to 10 on a dry weight basis.
The range is 0 g / l. Sources of carboxyl groups include gas, liquid, solid carbon dioxide and various carbonates such as potassium bicarbonate and potassium carbonate. The reaction temperature is from 10 to 40C, preferably from 20 to 30C, and the pH of the reaction is from 7.5 to 12, preferably from pH 8.5 to 10.
5, the pressure during the reaction can be from normal pressure to 100 kg / cm ² , and the reaction time is from 10 minutes to 100 hours.
Further, a reaction under CO ₂ supercritical conditions is also possible.

The desired polyhydric alcohol aromatic carboxylic acid can be separated from the reaction solution obtained as described above by purifying the reaction solution according to a conventional method. That is, the reaction solution is filtered, centrifuged, and the like to remove solids, and then the resulting solution is acidified by adding an acid solution such as hydrochloric acid or sulfuric acid, thereby obtaining a polyhydric alcohol aromatic. The carboxylic acid can be recovered. In addition, the target substance can be recovered by a method such as solvent extraction, and a known purification method such as chromatography can be appropriately used in combination.

In the production method of the present invention, the polyhydric alcohol aromatic carboxylic acid in the reaction solution can be appropriately detected and quantified by using high performance liquid chromatography. Specifically, analysis is performed by a general reverse phase chromatography using a silica gel packed column having an octadecyl group as a stationary phase and a mobile phase containing 0.1% trifluoroacetic acid added to a mixture of water and acetonitrile. Is possible. The detection is performed by a UV spectrophotometer at a wavelength around 280 nm.

The polyhydric alcohol aromatic carboxylic acid produced by the production method of the present invention is a compound represented by the general formula (2) (Formula 4), and specifically, α-resorcinic acid (resorcin-5) -Carboxylic acid), β-resorcinic acid (resorcin-4-carboxylic acid), γ-resorcinic acid (resorcin-2-carboxylic acid), resorcin-2,5
-Dicarboxylic acid, catechol-3-carboxylic acid, catechol-4-carboxylic acid, catechol-3,6-dicarboxylic acid, gentisic acid (hydroquinone carboxylic acid), hydroquinone-2,5-dicarboxylic acid, pyrogallol-4-
Examples thereof include carboxylic acid, pyrogallol-4,6-dicarboxylic acid, phloroglucin carboxylic acid, and phloroglucin dicarboxylic acid.

[0029]

The present invention will be described in detail with reference to typical examples, but the scope of the present invention is not limited to these examples. Example 1 Synthesis of γ-resorcinic acid using Propionibacterium freudenreich (1) A 21 mm diameter test of a normal broth medium (Eiken) (manufactured by Eiken Chemical Co., Ltd.) adjusted to a predetermined concentration. 5m to pipe
and autoclaved at 121 ° C. for 15 minutes. One platinum loop of the cells (Propionibacterium freudenreich MT-10883) maintained on a slant medium obtained by adding 1.5% agar to ordinary bouillon medium "Eiken" was inoculated into this. Shaking culture was performed in a constant temperature room at 25 ° C. for 2 days to grow the cells. A sufficiently grown culture solution.
5 ml was inoculated into 50 ml of a synthetic medium obtained by adding 20 mM to the composition shown in Table 1 (Table 1), followed by shaking at 25 ° C for 1 day. After completion of the culture, the culture was centrifuged at 3000 rpm for 10 minutes to recover the cells, and about 50 mg of cells were obtained in terms of dry weight.

[0030]

TABLE 1 _{────────────────────────────── NH 4 NO 3 0.1~10g / l} MgSO 4 · 7H 2 _{O 10~1000mg / l K 2 HPO 4} 25~2500mg / l NaH 2 PO 4 · 2H 2 O 5~2500mg / l CaCl 2 · 2H 2 O 5~500mg / l MnSO 4 · 4H 2 O 1~125mg / l _{ZnSO 4 · 7H 2 O 0.5~50mg /} l H 3 BO 3 0.5~50mg / l NiCl 2 · 6H 2 O 2~240μg / l Na 2 MoO 4 · 2H 2 O 12~1200μg / l CuSO 4 _{· 5H 2 O 1~125μg / l CoCl} 2 · 6H 2 O 1~125μg / l naFe-EDTA · 3H 2 O 2~210mg / l nicotinic acid 0.2~25mg / l glycine 0.1 to 10 mg / l hydrochloric acid Pyridoxine 25-25 0 Pg / l thiamine hydrochloride 25~2500μg / l folic acid 25~2500μg / l biotin 2~250μg / l pantothenate Ca 2~250μg / l vitamin B ₁₂ 2~250μg / l ──────────── ─────────────────

The obtained cells were resorcinol 1.10 mg.
(10 mM) 1 M carbonate buffer (pH 9.0) 1 m
and transferred to a 10 ml screw-type centrifuge tube.
After replacing the gas phase with 100% CO ₂ , the reaction was carried out while shaking in a constant temperature room at 25 ° C. for 3 days. After the reaction, 0.5
The reaction was quenched with 50 ml of ethanol-6N-HCl (50:50) solution and centrifuged (15000 rpm,
After 10 minutes), the supernatant was analyzed by high performance liquid chromatography under the following conditions. As a result, except for the resorcinol peak used as the raw material, the standard γ-
Only peaks consistent with resorcinic acid were detected. As a result of analyzing this peak by GC / MS, it was confirmed that the one showing the peak was γ-resorcinic acid. As a result of quantifying the production amount of γ-resorcinic acid, it was calculated that 0.51 mg was produced.

Column: Inertsil ODS-2 (GL Sc
ices Inc. Mobile phase: water: acetonitrile (85:15) Liquid sending: 0.8 ml / min Column temperature: 40 ° C. Detection method: UV absorption wavelength 280 nm

Example 2 Propionibacterium flo
Synthesis of γ-resorcinic acid using Idenreich (2) Culture grown in the same procedure as described in Example 1
1.10 mg of catechol was added to 50 ml of the cells equivalent to 50 ml of the solution.
Suspended in 1 ml of dissolved 1M carbonate buffer (pH 9.0)
And transferred to a test tube with a 10 ml screw cap.
After that, the gas phase _TwoAnd shake for 3 days.
Was. After the reaction, high-performance liquid chromatography was performed in the same manner as in Example 1.
When analyzed by the method using fee, 2,3-dihydrogen
A peak corresponding to roxybenzoic acid was observed.
The peak was analyzed by GC / MS.
It was confirmed to be dihydroxybenzoic acid. This method
As a result of the quantification by 2,3-dihydroxybenzoic acid
It was confirmed that 0.21 mg was produced.

[0034]

According to the present invention, it is possible to selectively produce a desired polyhydric alcohol aromatic carboxylic acid from an aromatic polyhydric alcohol under mild conditions of normal temperature and normal pressure. The method of the present invention is a simple and safe method as compared with the conventional chemical synthesis method, and is extremely useful as an industrial method for producing a polyhydric alcohol aromatic carboxylic acid.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) (C12N 1/20 C12R 1:01)

Claims (16)

[Claims] An animal or plant tissue or cell or microorganism having the ability to catalyze an aromatic polyhydric alcohol represented by the general formula (1) and a reaction represented by the reaction formula (1) (formula 1). Alternatively, the treated tissue, cells or microorganisms are brought into contact with an aqueous solvent in the presence of carbonate ions and / or CO ₂ , and a polyhydric alcohol aromatic represented by the general formula (2) is added to the reaction mixture. A method for producing a polyhydric alcohol aromatic carboxylic acid, which comprises producing a carboxylic acid and then separating the polyhydric alcohol aromatic carboxylic acid represented by the general formula (2) from the reaction system. Embedded image (Wherein, n represents 2 or 3, and m represents 1 or 2, respectively) 2. The method for producing a polyhydric alcohol aromatic carboxylic acid according to claim 1, wherein n in the reaction formula (1) is 2. 3. The method for producing a polyhydric alcohol aromatic carboxylic acid according to claim 2, wherein the aromatic polyhydric alcohol represented by the general formula (1) is resorcin or catechol. 4. The method for producing a polyhydric alcohol aromatic carboxylic acid according to claim 1, wherein m is 1 in the reaction formula (1). 5. The method according to claim 1, wherein the counter ion of the carbonate ion is selected from the group consisting of ammonia ion, various metal ions, bicarbonate ion which is an organic compound cation, and carbonate ion. The method for producing a polyhydric alcohol aromatic carboxylic acid according to claim 1. 6. The method for producing a polyhydric alcohol aromatic carboxylic acid according to claim 1, wherein the CO ₂ is a solid, a gas, or a supercritical fluid. 7. The method for producing a polyhydric alcohol aromatic carboxylic acid according to claim 1, wherein the cell or the cell is a tissue culture, a callus cell or a single cell. 8. The treated tissue, cell or microorganism is a crushed tissue, crushed cell, crushed cell, cell culture solution, cell culture solution, organic solvent-treated tissue, organic solvent-treated cell, organic solvent-treated cell, crude The method for producing a polyhydric alcohol aromatic carboxylic acid according to any one of claims 1 to 7, which is an enzyme or a purified enzyme. 9. The method for producing a polyhydric alcohol aromatic carboxylic acid according to claim 1, wherein the microorganism is a bacterium belonging to the genus Propionibacterium. 10. The bacterium belonging to the genus Propionibacterium is Propionibacterium freudenreich.
Onibacterium freudenreichii ), the method for producing a polyhydric alcohol aromatic carboxylic acid according to claim 9. 11. A tissue or cell or microorganism of an animal or a plant, or a treated product of the tissue, cell or microorganism, having an ability to act as a catalyst for the reaction represented by Reaction Formula (1). Embedded image (Wherein, n represents 2 or 3, and m represents 1 or 2, respectively) 12. The microorganism according to claim 11, which has an ability to act as a catalyst for the reaction represented by the reaction formula (1) or a treated product of the microorganism. 13. The microorganism according to claim 12, wherein the microorganism is a bacterium belonging to the genus Propionibacterium or a processed product of the microorganism. 14. The microorganism according to claim 13, wherein the bacterium is Propionibacterium freudenreichii or a processed product of the microorganism. 15. An enzyme which catalyzes the reaction represented by the reaction formula (1) derived from an animal, a plant or a microorganism having the ability to catalyze the reaction represented by the reaction formula (1). 16. The enzyme according to claim 15, wherein the enzyme is derived from Propionibacterium freudenreichii .