JP2005065554A - Method for producing 2,6-dihydroxybenzoic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing 2,6-dihydroxybenzoic acid, with which a bacterium having higher activity for converting resorcin to 2,6-dihydroxybenzoic acid is searched and which is industrially advantageously carried out by using the bacterium having the activity. <P>SOLUTION: A bacterium belonging to the genus Rhizobium is cultured by using a medium, resorcin is reacted with a carbonate ion and/or carbon dioxide in the presence of the obtained culture, treated material or immobilized material of the bacterium in an aqueous solvent to form 2,6-dihydroxybenzoic acid and 2,6-dihydroxybenzoic acid formed by the reaction is collected from the aqueous solvent. The method for producing 2,6-dihydroxybenzoic acid, with which the reaction selectivity of the bacterium is used and which is industrially advantageously carried out by an extremely simple process, comprises using the bacterium having higher activity for converting resorcin to 2,6-dihydroxybenzoic acid than a conventional bacterium. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for producing 2,6-dihydroxybenzoic acid used in pharmaceuticals and chemicals.

  2,6-dihydroxybenzoic acid is produced by a carbonation reaction by a Kolbe-Schmidt method using resorcin as a raw material. That is, it is produced by a method in which carbon dioxide gas is reacted with a metal salt of resorcin under high temperature and high pressure. According to this method, a mixture of the corresponding polyhydric alcohol aromatic carboxylic acids is generated, and from this mixture It is very difficult to separate and purify the target compound. For this reason, various methods for selectively producing the target compound have been tried, but have not yet been put to practical use.

  On the other hand, as a method for selectively introducing a carboxyl group at the 2-position of resorcinol, a method for producing 2,6-dihydroxybenzoic acid using the catalytic action of microbial cells is known (Japanese Patent Laid-Open No. 2001-46093). More specifically, as a microbial cell, a microorganism belonging to the genus Propionibacterium is used, and 2,6-dihydroxybenzoic acid is produced by contacting with resorcin in a carbonate buffer. .

  In addition, according to the first business book of the “Development of basic technology for production processes using biological functions” (interim evaluation) subcommittee (created by Biotechnology Development Office, New Energy and Industrial Technology Development Organization), Enterobacter The genus Agrobacterium is described as having a catalytic action to convert resorcin to 2,6-dihydroxybenzoic acid, but there is no further detailed description.

  As described above, as far as the present inventor is aware, there are limited reports on microorganisms having a catalytic action for converting resorcin to 2,6-dihydroxybenzoic acid.

  In order to industrially produce industrially useful substances using the catalytic action of microbial cells, it is important to reduce the production cost of the microbial cells in the total production cost of the useful materials, More specifically, it is necessary to increase the production rate of the useful substance per unit microorganism weight / unit time (hereinafter referred to as cell activity). As one of the methods for this purpose, a method of enhancing the target catalytic action by adding an appropriate chemical substance generally called an inducer is generally well known when cultivating the microorganism and preparing cells. It has been.

  For example, in Japanese Patent Application Laid-Open No. 2001-46093, adding 2,6-dihydroxybenzoic acid to a medium for growing bacterial cells and culturing enhances the activity of converting resorcin to 2,6-dihydroxybenzoic acid. It is stated that it is effective. However, even in such a case, as shown in a comparative example described later, the cell activity for converting resorcinol to 2,6-dihydroxybenzoic acid is not always sufficient. In the same report, there is no disclosure of the effect of 2,6-dihydroxybenzoic acid on microorganisms belonging to the genus Rhizobium.

On the other hand, Tschech et al. (Arch. Microbiol., 155: 68-74 (1990)) added 2,6-dihydroxybenzoic acid to a medium and cultured in a mixed culture of the genus Clostridium. -Although it has been reported that the activity of converting dihydroxybenzoic acid to resorcin is induced, there is no disclosure about the conversion of resorcin to 2,6-dihydroxybenzoic acid.
That is, the measures for enhancing the catalytic action of converting resorcin possessed by microbial cells into 2,6-dihydroxybenzoic acid are limited, and the activity is not sufficient.

Japanese Patent Laid-Open No. 2001-46093 Business book for the first “Development of basic technology for production processes using biological functions” (interim evaluation) subcommittee (created by Biotechnology Development Office, New Energy and Industrial Technology Development Organization): HP address http: // www. nedo.go.jp/iinkai/hyouka/nittei/150512.html Arch. Microbiol., 155: 68-74 (1990)

  The present invention searches for microorganisms having a higher activity for converting resorcinol to 2,6-dihydroxybenzoic acid, and is advantageous for industrial implementation using microorganisms having these activities. The manufacturing method of this is provided.

  As a result of intensive studies on a method for producing 2,6-dihydroxybenzoic acid in order to solve the above-mentioned problems, the present inventors have newly discovered that a microorganism belonging to Rhizobium has been released from resorcin from 2,6-dihydroxybenzoic acid. The present inventors have found that it has an activity to convert to an acid, and have completed the present invention.

  In particular, the Rhizobium sp MTP-10005 strain found by the present inventor has been found to have a remarkably high activity for converting resorcin to 2,6-dihydroxybenzoic acid.

  Furthermore, surprisingly, when cultivating a microorganism belonging to Rhizobium, a microorganism having a higher cell activity than conventionally known microorganisms can be obtained simply by the presence of 2,6-dihydroxybenzoic acid in the medium. At the same time, it was found that a body could be prepared, and the present invention was completed.

That is, the present invention provides a microorganism having a higher activity to convert resorcin to 2,6-dihydroxybenzoic acid than conventionally known microorganisms, and therefore, production of 2,6-dihydroxybenzoic acid which is industrially advantageous. The law is provided, and more specifically, as follows.
[1] A microorganism belonging to the genus Rhizobium is cultured in a medium, and carbonate ions and / or carbon dioxide are resorcinated in an aqueous solvent in the presence of the culture, treatment or immobilization product of the obtained microorganism. 2,6-dihydroxybenzoic acid produced by reaction with 2,6-dihydroxybenzoic acid produced by the reaction is collected from the aqueous solvent.
[2] The 2,6-dihydroxybenzoic acid according to [1], wherein the microorganism belonging to the genus Rhizobium is a microorganism belonging to the genus Rhizobium that does not include the genus Agrobacterium. Production method.
[3] The microorganism according to [1] or [2], wherein the microorganism belonging to the genus Rhizobium is a microorganism having a base sequence on the 5 ′ end side of 16S rDNA of the sequence shown in SEQ ID NO: 1 in the Sequence Listing. , 6-Dihydroxybenzoic acid production method.
[4] The microorganism according to any one of [1] to [3], wherein the microorganism belonging to the genus Rhizobium is a microorganism belonging to Rhizobium sp MTP-10005 (FERM P-19426). , 6-Dihydroxybenzoic acid production method.
[5] When culturing a microorganism belonging to the genus Rhizobium, 2,6-dihydroxybenzoic acid is added to the medium, 2 according to any one of [1] to [4] , 6-Dihydroxybenzoic acid production method.

  According to the present invention, by using a microorganism having a higher activity to convert resorcin to 2,6-dihydroxybenzoic acid than in the past, its reaction selectivity is also utilized, which is advantageous for industrial implementation in a very simple process. A process for producing 6-dihydroxybenzoic acid is provided.

  The microorganism in the present invention may be any microorganism as long as it belongs to the genus Rhizobium, and more specifically, Rhizobium sp MTP-10005 (FERM P-19426) is a preferred example. I can list them. This strain is a strain isolated from the soil of Suita City, and the bacteriological properties of this strain are as shown in [Table 1] below.

Based on the above mycological characteristics, “Bergey's Manual of Systematic Bacteriology Vol. 1 (1984) Williams & Wilkins”, “Bergey's Manual of Systematic Bacteriology Vol.1 (1984) Williams & Wilkins” G I Barrow
& Earl Kay Aye Feltham version:. Cohen and steel scan Manual For The identification of medical bacteria, Third Edition Cambridge University Press (1993) [GI Barrow and RKAFeltham ed, Cowan &Steel's Mannual for the Identification of Medical Bacteria 3 rd .ed , Cambridge univ.press, (1993)], and as a result of identification of the MTP-10005 strain, it was shown that this strain has similar properties to Rhizobium radiobacter . However, it did not assimilate potassium gluconate and did not completely match the typical properties of Rhizobium radiobacter.

  Furthermore, after smearing this strain on LB Broth (Becton Dickinson) agar medium, genomic DNA was extracted from the cells obtained by culturing at 30 ° C. for 2 days using PrepMan Method (Applied Biosystem). The extracted genomic DNA was used as a template to amplify a region of about 500 bp at the 5 ′ end of 16S rDNA by PCR. The amplified base sequence was sequenced, and the base sequence of 16S rDNA was obtained as shown in SEQ ID NO: 1 in the Sequence Listing.

  As a result of analyzing the partial base sequence of 16S rDNA determined as described above using homology search software, the homology rate of Agrobacterium rubi, that is, the current name Rhizobium rubi, is 97.2%. However, no reference strain was found that found a high homology of 99.0% or higher where species identity was discussed. The homology with Rhizobium radiobacter was 97.0%.

  For purification of PCR products and cycle sequencing, MicroSeq500 16S rDNA Bacterial Sequencing Kit (Applied Biosystem) was used. For the homology analysis, Microseq Microbial Identification System Software V.1.4.1 (Applied Biosystem) was used, and Microseq Microbial 500 Library v.0023 was used as a database for homology search.

According to Young et al., It is proposed that the genus Agrobacterium is included in the genus Rhizobium (International Journal of Systematic
and Evolutionary Microbiology (2001), 51, 89-103). From the above bacteriological properties and 16S rDNA partial base sequence analysis, this strain does not belong to any species of the genus Rhizobium or Agrobacterium. Based on the above facts, this strain was named Rhizobium sp MTP-10005. This strain has been deposited under the accession number FERM P-19426 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 1-1-1, Higashi, Ibaraki Prefecture, from July 8, 1993 .

  Representative examples of the microorganisms used in the present invention are as described above, but in the present invention, also for strains in which the catalytic activity of 2,6-dihydroxybenzoic acid is enhanced by mutation treatment or the like for the same strains, It is included in the scope of the invention. Examples of such mutant strains include mutant strains having improved resistance to resorcin and 2,6-dihydroxybenzoic acid, mutant strains that no longer decompose resorcin, 2,6-dihydroxybenzoic acid, and 2,6-dihydroxybenzoic acid. Examples include mutants with high productivity of enzymes involved in benzoic acid.

  The microorganism culture referred to in the present invention can be obtained by adopting a method based on a general microorganism cultivation method in the field, and more specifically, the conditions are as follows.

  The culture medium is a medium in which the microorganism is usually used in this field, for example, organic nutrient sources such as meat extract, yeast extract, malt extract, peptone, NZ amine, carbon such as glucose, maltose, sucrose, starch, organic acid, etc. Sources, nitrogen sources such as ammonium sulfate, urea, ammonium chloride, inorganic nutrient sources such as phosphate, magnesium, potassium, iron, and vitamins may be used in appropriate combination.

  The culture temperature may be 20 to 40 ° C., more preferably 25 to 35 ° C., and the culture pH may be pH 5.0 to 10.0, more preferably pH 6.0 to 9.0. Moreover, although it can be performed under any condition under anaerobic and aerobic conditions, usually, shaking culture under aerobic conditions with a high growth rate is preferred. The number of days of culture is not particularly limited, but it may be cultured until the desired activity is maximized in the range of 1 to 7 days.

  In order to increase the activity of converting resorcin to 2,6-dihydroxybenzoic acid, it is effective to add 2,6-dihydroxybenzoic acid to the medium for growing the cells. The addition amount is preferably 1 to 100 mM, preferably 10 to 50 mM, in the medium.

  The above culture conditions can be changed according to the microorganism used, the composition of the medium, etc., but it is important to set the maximum activity for converting resorcin to 2,6-dihydroxybenzoic acid. It is natural to be.

  The microorganism culture referred to in the present invention includes the culture solution itself obtained by culturing microorganisms as described above, or the microorganism cells separated from the culture solution by operations such as centrifugation and filtration. .

  The treated product of microorganisms as used in the present invention refers to a washed product, crushed product, or extract of the above-mentioned microbial cells. The washed cells are obtained by washing the cells with physiological saline, and the dried cells are obtained by freeze-drying or acetone-drying the cells or washed cells. The crushed cell body can be obtained by various physicochemical methods such as ultrasonic crushing, French press, osmotic pressure, freeze-thawing, lytic enzyme, surfactant and organic solvent treatment. Extracts, for example, from microbial cell disruptions and culture supernatants, by conventional methods such as ammonium sulfate fractionation, ion exchange chromatography, gel filtration, hydrophobic chromatography, affinity chromatography (including combinations thereof), An active fraction that converts resorcin to 2,6-dihydroxybenzoic acid can be identified and separated.

The microorganism-immobilized product as referred to in the present invention is a known method such as polyacrylamide method, alginic acid method, carrageenan method, or covalent bonding method, adsorption method, etc. It can be obtained by immobilization.

  These microorganism cultures, processed products, and immobilization products can be used by reacting immediately after their production, as well as stored temporarily after production, for example, under low-temperature conditions, and used as necessary. You can also.

  The microbial culture / processed / immobilized product thus prepared is allowed to act on resorcin in an aqueous solvent in order to produce 2,6-dihydroxybenzoic acid. The amount of the compound used is usually an amount that exhibits the intended effect (effective amount), and this effective amount can be easily determined by a person skilled in the art by a simple preliminary experiment. For example, if washed wet cells are used, 10 to 40 g per liter of reaction solution is one standard.

  The aqueous solvent in the present invention may be a homogeneous system composed of water or a mixture of water and a solvent miscible with water, as long as it does not adversely affect the production of 2,6-dihydroxybenzoic acid, or water and water. It may be a two-phase system comprising a solvent immiscible with water or a mixture of water and water miscible solvent and water immiscible solvent.

The presence of carbonate ions and / or carbon dioxide in the present invention is to allow a carboxyl group source to be added to resorcin to coexist in an aquatic solvent, and more specifically, sodium bicarbonate and sodium carbonate. It can be prepared by adding a representative carbonate to the above-mentioned aquatic solvent, or blowing or adding gaseous, liquid, or solid carbon dioxide into the aquatic solvent. The concentration of carbonate ions and / or carbon dioxide in the aqueous medium is not particularly limited, but a concentration of 50 to 5000 mM can be exemplified. Further, the reaction under CO ₂ supercritical conditions is not particularly limited.

The conditions for producing 2,6-dihydroxybenzoic acid in the present invention (hereinafter simply referred to as reaction) are pH 3-12, preferably pH 6-9, as the reaction pH. Further, when the pH of the reaction solution changes due to the progress of the reaction, it is preferable to adjust the pH to the optimum pH. The reaction temperature is in the range of freezing point to 60 ° C, preferably 20 to 40 ° C. The reaction time is not particularly limited, but more specifically is about 1 minute to 120 hours. The pressure during the reaction is not particularly limited, but usually a range of normal pressure to 100 kg / cm ² is suitable. The resorcin concentration during the reaction is not particularly limited, but is preferably 1 to 2000 mM. Resorcin may be added in the form of a solid, dissolved in water or an organic solvent that does not affect the reaction, or dispersed in a solvent containing an appropriate surfactant. Moreover, it may be added all at once at the beginning of the reaction, or a required amount may be added in divided portions as the reaction proceeds. Furthermore, the above reaction method may be either under stirring or standing, and may be either batch type or continuous type using an appropriate bioreactor.

  The method for collecting 2,6-dihydroxybenzoic acid from the aqueous solvent in the present invention is not particularly limited as long as it is a method for separating and purifying the produced 2,6-dihydroxybenzoic acid from the reaction solution. A method of recovering crude crystals of 2,6-dihydroxybenzoic acid by pretreating the reaction solution by filtration, centrifugation, etc., and then acidifying the resulting upper semen by adding an acid solution such as hydrochloric acid or sulfuric acid Can be cited as a suitable example. Further, it can be recovered by a method such as solvent extraction, and a known purification method such as chromatography can be used in combination as appropriate.

In the production method of the present invention, detection and quantification of 2,6-dihydroxybenzoic acid are performed, for example, by high performance liquid chromatography. That is, a silica gel packed column having an octadecyl group is used as a stationary phase, and 0.1% in a mixture of water and acetonitrile.
Analysis can be performed by general reverse phase chromatography using a trifluoroacetic acid added as a mobile phase. The detection is performed in the vicinity of a wavelength of 280 nm by an ultraviolet partial light detector.

  EXAMPLES Next, although an Example demonstrates this invention still in detail, this invention is not limited to these Examples.

  5 ml of LB medium (yeast extract: 5 g / l, polypeptone: 10 g / l, NaCl: 10 g / l pH 7.0) adjusted to a predetermined concentration is placed in a test tube with a diameter of 21 mm and sterilized at 121 ° C. for 15 minutes Went. One platinum loop was inoculated with cells (Rhizobium sp. MTP-10005) subcultured in a slant medium in which 1.5% agar was added to LB medium. Shaking culture was performed in a thermostatic chamber at 25 ° C. for 1 day to grow seed cells. 0.5 ml of the mixture was inoculated into a 500 ml baffled Erlenmeyer flask containing 50 ml of an autoclave-sterilized medium having a composition shown in Table 2 (adjusted to pH 8.5 with 5N KOH). The cells were cultured with shaking at 25 ° C. for 2 days, and the cells were collected by centrifugation (at 3000 rpm for 10 minutes) (about 50 mg in terms of dry weight).

  The collected cells were washed with physiological saline (NaCl 9 g / l) and then suspended in 10 mM potassium phosphate buffer (pH 7.0) containing 0.02% 2-mercaptoethanol in 1/20 of the culture solution. . The suspension was crushed by ultrasonic treatment, and after centrifugation (15000 rpm, 10 minutes), the supernatant was collected and used as an enzyme solution. Next, 1 ml of a reaction solution having the reaction composition shown in Table 3 below was reacted in a 10 ml screw-centrifuge centrifuge tube while shaking at 30 ° C. for 5 minutes. The reaction was stopped by adding 200 μl of 5N hydrochloric acid.

After deproteinization by centrifugation (15000 rpm, 10 minutes), the supernatant was analyzed by high performance liquid chromatography under the following conditions. Analyzed by HPLC.
As a result, only peaks corresponding to 2,6-dihydroxybenzoic acid of the sample were detected except for the resorcin peak used as a raw material. As a result of analyzing this peak by LC / MS, it was confirmed to be 2,6-dihydroxybenzoic acid.

Column: Inertsil ODS-2 (manufactured by GL Sciences Inc.)
Mobile phase: water: acetonitrile (85:15)
Liquid feed: 0.6 ml / min
Column temperature: 40 ° C
Detection method: UV absorption wavelength 280 nm
As a result of quantifying the amount of 2,6-dihydroxybenzoic acid produced by this method, it was calculated that 3 μg was produced.

  In the method described in Example 1, the same operation was performed except that 2,6-dihydroxybenzoic acid was added to the medium so as to have a concentration of 20 mM, and the amount of 2,6-dihydroxybenzoic acid produced in the reaction was measured. As a result, the amount of cells obtained was equivalent to Example 1 (about 50 mg in terms of dry weight), but the amount of 2,6-dihydroxybenzoic acid produced by the reaction was calculated to be 60 μg. It was.

[Comparative Example 1]
Propionibacterium freudenreichii disclosed in JP 2001-46093 A
MT-10883 (FERM P-17415) was cultured and reacted in the same manner as in Example 1, and the amount of 2,6-dihydroxybenzoic acid produced was quantified. As a result, the amount of cells obtained was the same as in Example 1 ( The amount of 2,6-dihydroxybenzoic acid produced by the reaction was calculated to be 1 μg.

[Comparative Example 2]
Propionibacterium freudenreichii MT-10883 (FERM P-17415) disclosed in JP-A-2001-46093 was cultured and reacted in the same manner as in Example 2, and the amount of 2,6-dihydroxybenzoic acid produced was quantified. The amount of the obtained cells was the same as in Example 1 (about 50 mg in terms of dry weight), but the amount of 2,6-dihydroxybenzoic acid generated by the reaction was calculated to be 8 μg.

Claims (5)

  A microorganism belonging to the genus Rhizobium is cultured in a medium, and carbonate ions and / or carbon dioxide are allowed to act on resorcin in an aqueous solvent in the presence of a culture, treatment or immobilization product of the obtained microorganism. 2,6-dihydroxybenzoic acid is produced, and 2,6-dihydroxybenzoic acid produced by the reaction is collected from the aqueous solvent.   The method for producing 2,6-dihydroxybenzoic acid according to claim 1, wherein the microorganism belonging to the genus Rhizobium is a microorganism belonging to the genus Rhizobium that does not contain the genus Agrobacterium.   The 2,6-dihydroxybenzoic acid according to claim 1 or 2, wherein the microorganism belonging to the genus Rhizobium is a microorganism having a base sequence on the 5 'end side of 16S rDNA of the sequence shown in SEQ ID NO: 1 in the sequence listing. Acid production method.   The 2,6-dihydroxybenzoic acid according to any one of claims 1 to 3, wherein the microorganism belonging to the genus Rhizobium is a microorganism belonging to Rhizobium sp MTP-10005 (FERM P-19426). Acid production method. The 2,6-dihydroxybenzoic acid according to any one of claims 1 to 4, wherein 2,6-dihydroxybenzoic acid is added to the medium when culturing a microorganism belonging to the genus Rhizobium. Acid production method.