JPH0779768A - Novel microorganism and production of 2,6-naphthalenedicarboxylic acid with the microorganism - Google Patents

Abstract

PURPOSE:To efficiently obtain 2,6-naphthalendicarboxylic acid by culturing a new microorganism of the genus Sphingomonas in an organic solvent medium comprising a culture medium and an organic solvent containing 2,6- dimethylnaphthalene dissolved therein. CONSTITUTION:Sphingomonas paucimobilis A-7 strain (FERM P-13632) is cultured in an organic solvent medium comprising a culture medium and an organic solvent containing 2,6-dimethylnaphthalene dissolved therein. Both the methyl groups of the 2,6-dimethylnaphthalene dissolved in the organic solvent are efficiently oxidized. The conversion substrate in the solvent as well as the culture medium can efficiently be supplied.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention oxidizes 2,6-dimethylnaphthalene using a novel microorganism belonging to the genus Sphingomonas and an enzyme derived from the resting microorganism or the microorganism of the microorganism,
It relates to a method for producing 2,6-naphthalenedicarboxylic acid.

[0002]

2. Description of the Related Art 2,6-naphthalenedicarboxylic acid or its ester derivative is a compound useful as a high-performance resin raw material, a liquid crystal raw material, a polyamide-based pharmaceutical raw material, and a dye / pigment. Being produced. In particular, the usage as polyethylene naphthalate (PEN) resin is said to be 80 to 90%. Compared to the current polyethylene terephthalate (PET) resin, the heat resistant temperature is about 35 ° C and the breaking strength is also about 25% higher. Mass production is expected as a material having excellent properties with respect to the second-order transition point, crystallization rate, softening point, melt viscosity, and the like.
However, the chemical synthesis method using 2,6-dimethylnaphthalene as a raw material is a high-temperature reaction, so that functional group transfer is likely to occur, and it is difficult to obtain high-purity 2,6-naphthalenedicarboxylic acid. There was also a problem that it was necessary, consumed a large amount of energy, and polluted the environment.

As a method for solving these problems, research on a microbial oxidation method using a microorganism has been advanced in recent years. The oxidation method using a microorganism as a catalyst has an excellent feature that it can be reacted at room temperature and atmospheric pressure, and that functional groups are not transferred, so that by-products are hardly generated. However, according to the reports so far, even if 2,6-dimethylnaphthalene is oxidized using a microorganism such as Flavobacterium, only one methyl group is oxidized, and 2,6
-Naphthalenedicarboxylic acid was not detected (E.
A. BARNSLEY APPLIED AND EN
VIRONMENTAL MICROBIOLOGY
54, 428-433, 1988).

A report on confirmation of 2,6-naphthalenedicarboxylic acid by co-oxidation with sugars by Nocardia bacterium (G.K.SKRYABIN, et al., D
OKL. AKAD. NAUK. USSR 202,97
3 to 974, 1972), but the yield was low and the study was qualitative.

More recently, a method for producing 2,6-naphthalenedicarboxylic acid by a fermentation method using Pseudomonas bacteria (Japanese Patent Laid-Open No. 3-80091) and an oxidizing power for an alkyl group of an alkylnaphthalene compound have been developed. A method for producing naphthalenecarboxylic acid using microorganisms (Japanese Patent Laid-Open No. 5-15365) has been proposed.

[0006]

However, although the above-mentioned method is preferable as an energy-saving and environmental conservation type production method, the culture conditions for the production are aqueous, and in addition, as a conversion substrate, 6-Dimethylnaphthalene is insoluble in water and is a so-called solid-liquid reaction system, so that the supply amount of the conversion substrate is limited, and it is difficult to control the supply amount, and in terms of efficient continuous mass production, It contains a problem.

Therefore, a microorganism having an ability to oxidize both methyl groups of 2,6-dimethylnaphthalene, capable of growing or reacting even in an organic solvent system, and not inhibiting the oxidation reaction was found. If an organic solvent in which 2,6-dimethylnaphthalene is dissolved can be supplied to such a microorganism, the liquid-liquid reaction system can continuously provide 2,
Since large-scale conversion to 6-naphthalenedicarboxylic acid is also possible and it is highly useful for the petroleum industry and petrochemical industry, it has been desired to establish a bacterium that can be used in such a reaction system and a production method.

[0008]

Under these circumstances, the present inventors have conducted extensive studies in order to solve the above-mentioned problems, developed an effective screening method for bacterial strains, and carried out efficient search for microorganisms. As a result, a novel microorganism capable of efficiently oxidizing the methyl groups on both sides of 2,6-dimethylnaphthalene dissolved in an organic solvent was found, and a production method using a liquid-liquid reaction system was made possible for the first time to complete the present invention.

That is, the present invention relates to a 2,6-naphthalenedicarboxylic acid-producing bacterium belonging to the genus Sphingomonas and produced from 2,6-dimethylnaphthalene dissolved in an organic solvent.
A method for producing 2,6-naphthalenedicarboxylic acid, which comprises culturing and converting in a liquid-liquid system consisting of an organic solvent in which 6-dimethylnaphthalene is dissolved and a medium, that is, an organic solvent system, and 2,6- 2,6-naphthalenedicarboxylic acid which is obtained by contacting an immobilized bacterial cell of a naphthalenedicarboxylic acid-producing bacterium or an enzyme derived from the bacterial cell with 2,6-dimethylnaphthalene dissolved in an organic solvent in an organic solvent system for conversion. A method for producing an acid is provided.

The novel microorganism of the present invention has a carbon source of 2,
It consists of a medium that grows in a medium containing only 6-dimethylnaphthalene and has the ability to produce 2,6-naphthalenedicarboxylic acid, or one in which only 2,6-dimethylnaphthalene is dissolved in an organic solvent. It can be isolated by growing it in an organic solvent-based culture and screening for its ability to produce 2,6-naphthalenedicarboxylic acid.

Among soils collected by the inventors from all over the country, among the components necessary for the growth of microorganisms, a medium containing no carbon source (hereinafter referred to as medium) was dispensed into a test tube or the like and sterilized. After that, soil is added, and an organic solvent in which 2,6-dimethylnaphthalene or 2,6-dimethylnaphthalene is dissolved, such as decalin, is added, and culturing is performed by a test tube shaker or the like. This culture solution is subcultured in a similar medium previously dispensed into another test tube or the like using a platinum loop or the like, then 2,6-dimethylnaphthalene is added, and the test tube shaker or the like is used. Incubate further. After culturing, the culture solution is diluted with the above-mentioned medium or sterilized water and applied to an agar medium containing no carbon source, and then 2,6-dimethylnaphthalene is supplied to further culture. The 2,6-naphthalenedicarboxylic acid-producing bacterium can be selected by isolating the colonies obtained by the culture and confirming the productivity of each strain for producing 2,6-naphthalenedicarboxylic acid.

As the medium for culturing these bacteria, general medium components other than the carbon source can be used. That is, as the nitrogen source, for example, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium carbonate, ammonium acetate, ammonium nitrate, sodium nitrate, urea and the like, and as inorganic salts, for example, potassium,
Various salts such as sodium, iron, magnesium, manganese, copper and calcium can be used. The culture conditions are
Generally, culture conditions may be such that microorganisms are not killed. For example, the culture is carried out aerobically at a pH of about 5-9 and a temperature of about 20-40 ° C.

The production capacity of the obtained strains for 2,6-naphthalenedicarboxylic acid was confirmed in a medium containing 2,6-dimethylnaphthalene as the carbon source of each strain.
After separating the culture cultivated under the same conditions as above by centrifugation or the like, the supernatant is subjected to an appropriate analytical method, for example, high performance liquid chromatography (HPLC), gas chromatography (GC), nuclear magnetic resonance spectrum (NMR). , Infrared absorption spectrum (IR), ultraviolet absorption spectrum (UV)
Etc. may be used for analysis. The obtained microorganisms had the mycological properties shown in Table 1 below as a result of testing using API20NE (Biomerie) for identifying non-fermentative bacteria other than enterobacteria.

[0014]

[Table 1]

Based on the mycological properties shown in Table 1, BERGE
Y'S MANUAL OF SYSTEM
BACTERIOLOGY, vol. 1, P198 (1
984), INTERNATIONAL JOURNA
L OF SYSTEMATIC BACTERIOL
OGY, vol. 40, No. 3, P320-321
(1990), when classified by the API20NE profile index, it was recognized as belonging to Sphingomonas paucimobilis.

The present bacterium can grow in an organic solvent system using 2,6-dimethylnaphthalene as a single carbon source, and accumulates 2,6-naphthalenedicarboxylic acid in the medium. No 2,6-naphthalenedicarboxylic acid-producing bacterium having such characteristics has been reported yet.

Based on the above-mentioned mycological properties, the present inventors determined that the bacterium is a new strain belonging to Sphingomonas paucimobilis, and determined that the bacterium is Sphingomonas paucimobilis A-7 (Sphingomonas paucimobilis).
bilis A-7) strain and deposited at the Institute of Biotechnology, Institute of Industrial Science [FERM P-1363].
2].

The microorganism of the present invention is, in addition to the microorganism belonging to Sphingomonas pautimobilis and capable of oxidizing the methyl group at the 2,6-position, such microorganisms as, for example, X-ray / ultraviolet irradiation, ethyl methane sulfonic acid, etc. It also includes the use of so-called mutant strains that have been subjected to known mutation treatment such as treatment with a mutagen, and the use of breeding strains by genetic engineering and the like.

Next, the method for producing 2,6-naphthalenedicarboxylic acid according to the present invention will be described by taking as an example the case where Sphingomonas pautimobilis A-7 strain (hereinafter referred to as the present bacterium) is used.

The medium components for culturing the bacterium include:
Other than containing 2,6-dimethylnaphthalene as a carbon source and a conversion substrate, other medium components are not particularly limited as long as the growth of the bacterium is good. That is, as a growth substrate, for a nitrogen source, for example, ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium carbonate, ammonium acetate, ammonium nitrate, sodium nitrate, urea and the like, as inorganic salts, for example, potassium, sodium, iron, magnesium, Various salts such as manganese, copper, calcium and cobalt can be used. In addition to 2,6-dimethylnaphthalene, salicylic acid, 2-methylnaphthalene, anthracene, glucose, fructose, starch and the like can be added as a carbon source as an auxiliary substrate.

Examples of the solvent for dissolving 2,6-dimethylnaphthalene used in the organic solvent culture include heptane, n-octane, isooctane, cyclooctane, nonane, decane, dodecane, tetralin, decalin, hexyl ether and the like. Although it can be used, it does not decrease in volatilization after culturing for several days, and it is neither too weak nor too weak in microbial toxicity in the sense that it suppresses the growth of miscellaneous bacteria.
Solvents other than the above can also be used as long as they dissolve dimethylnaphthalene and are not assimilated. And
Any one of these may be used, or two or more of them may be arbitrarily mixed and used.

The method of dissolving and supplying 2,6-dimethylnaphthalene in a solvent as in the present invention, compared with the solid-liquid reaction method of directly supplying 2,6-dimethylnaphthalene to an aqueous medium, Since the dispersibility of the conversion substrate is extremely good, it becomes easy to contact with the bacterial cells, and since the conversion substrate in the solvent is easy to be in a uniform state, the supply amount can be easily controlled, and not only the medium The conversion substrate in the solvent can be efficiently and continuously or intermittently and freely supplied, so that a continuous reaction is possible and the production efficiency of 2,6-naphthalenedicarboxylic acid can be further enhanced. Further, the present bacterium can produce 2,6-naphthalenedicarboxylic acid by directly supplying it without dissolving 2,6-dimethylnaphthalene in a solvent as in the conventional method, but its production efficiency is naturally poor. Will be things.

The culture conditions may be any as long as the bacterium can proliferate without being killed. For example, the culture temperature is about 15 to 37 ° C, more preferably about 25 to 35 ° C, and the pH of the medium is about 4.2 to 8.
9, more preferably in the range of 6.0-8.0, about 1-3.
It is preferable to culture or react aerobically for 0 days.

In order to efficiently produce the desired 2,6-naphthalenedicarboxylic acid by culturing the bacterium using 2,6-dimethylnaphthalene as a single carbon source or conversion substrate, 2,6-dimethylnaphthalene is used. It is necessary to add at least an amount necessary for the present bacterium to grow. Since this bacterium is hardly affected by 2,6-dimethylnaphthalene, it is possible to add a large amount of this, but when the amount of 2,6-naphthalenedicarboxylic acid produced increases, Since the pH will drop, supply an alkaline solution such as sodium hydroxide, ammonia water, potassium hydroxide, etc., supply a component with a buffering capacity, or use it in the medium beforehand to adjust the pH to an appropriate range. By doing so, more efficient production can be achieved. Further, the cells may be cultured and proliferated in advance under the above-mentioned conditions to collect the cells, and then the cells may be subjected to the method described below.

The process of producing 2,6-naphthalenedicarboxylic acid from 2,6-dimethylnaphthalene using the present bacterium may be a batch type process or a bioreactor or the like. In addition, the cells of the present bacterium, a treated product thereof, or an enzyme can also be immobilized according to a conventional immobilization method using, for example, calcium alginate, polyacrylamide method, polyurethane resin method, photocrosslinkable resin method, or the like. The obtained bacterial cell culture can be used as it is as an enzyme source, but it is preferable to use microbial cells obtained by solid-liquid separation by an operation such as centrifugation. Further, the microbial cells may be washed with a solution such as a phosphate buffer solution and suspended in the solution before use. In addition, the enzyme derived from the microbial cell can be purified by a conventional method, and when it is produced by using these resting microbial cell, treated product of the microbial cell or enzyme, it can be carried out under the above-mentioned culture conditions. The 2,6-naphthalenedicarboxylic acid in the culture solution or reaction solution may be purified by a conventional method. That is, the 2,6-naphthalenedicarboxylic acid is recovered by treating the culture solution or the reaction solution by filtration, centrifugation, etc., and acidifying the resulting solution with an acid solution such as hydrochloric acid or sulfuric acid. You can It is also possible to recover by a method such as solvent extraction, and a known purification method such as chromatography can be appropriately used in combination.

The production method of the present invention was made possible for the first time by finding a microorganism capable of efficiently oxidizing the methyl groups on both sides of 2,6-dimethylnaphthalene dissolved in an organic solvent.

[0027]

The present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples.

[0028]

Example 1 From 2,6-dimethylnaphthalene to 2,6-
In order to isolate bacteria that produce naphthalenedicarboxylic acid, screening was performed using a medium containing the components shown in Table 2 dissolved in deionized water. The pH of this medium became 7.

[0029]

[Table 2]

100 ml of this medium was placed in a 500 ml Erlenmeyer flask with baffles and sterilized at 121 ° C. for 20 minutes. After cooling at room temperature, add 1-2 scoops of soil collected in various places, add 100 mg of separately sterilized 2,6-dimethylnaphthalene, and 30 ° C., 270 rpm
The cells were shaken and cultured. After inoculating 0.1 ml of another similar medium, shake culture was carried out again at 30 ° C. and 270 rpm, and after one week, a part of the culture solution was diluted with sterilized physiological saline and adjusted in advance. The agar medium containing the components shown in Table 2 was applied to a plate medium containing agar at a concentration of 15 g / l, and a diethyl ether solution in which 2,6-dimethylnaphthalene was dissolved was sprayed and supplied onto the plate medium. , 30
The cells were cultured at 0 ° C for 1 week.

Colonies having zona pellucida formed on the plate medium and showing assimilation of 2,6-dimethylnaphthalene were isolated, and all of these microorganisms were subjected to 2,6 by the method described below. -A naphthalene dicarboxylic acid producing strain was selected. That is, 4 ml of the same medium as above, 2,6
-Decalin 4 with dimethylnaphthalene dissolved in 1 wt%
Dispense ml into a test tube with an inner diameter of 21 mm, and
After sterilizing for 0 minutes and cooling at room temperature, 1 platinum loop of the isolated microorganism was planted using a platinum loop. Reciprocal shaking culture was carried out at 30 ° C. and 300 pm for several days using a test tube shaker. After culturing, the supernatant of the aqueous portion of this culture broth was treated with a strong alkali and then neutralized by high performance liquid chromatography (HPL
C.), a plurality of 2,6-naphthalenedicarboxylic acid-producing bacteria were selected, and among them, Sphingomonas pautimobilis A-7 strain having the mycological properties shown in Table 1 was obtained.

[0032]

Example 2 In a 500 ml Erlenmeyer flask with a baffle, 50 ml of the same medium as in Example 1 and 50 ml of decalin in which 2,6-dimethylnaphthalene was dissolved in 1 wt% were placed and sterilized at 121 ° C. for 20 minutes. After cooling at room temperature, one platinum loop of the Sphingomonas pautimobilis A-7 strain obtained in Example 1 was inoculated and shake-cultured at 30 ° C. and 300 rpm for 4 days. After culturing, the supernatant of the aqueous portion of the culture broth was analyzed in the same manner as in Example 1 to measure the production amount of 2,6-naphthalenedicarboxylic acid. The results are shown in Table 3.

[0033]

Comparative Example 1 The same medium as in Example 1 was used except that the method for supplying 2,6-dimethylnaphthalene in Example 2 was changed to a conventional solid-liquid reaction system in which the 2,6-dimethylnaphthalene was directly added to the medium without being dissolved in a solvent. Was cultured for 4 days under the same conditions as in Example 2 and analyzed to measure the production amount of 2,6-naphthalenedicarboxylic acid. 2,6-dimethylnaphthalene is 500m
g was added. The results are shown in Table 3. The results in Table 3 show that the method using the organic solvent system of the present invention is superior.

[0034]

[Table 3]

[0035]

Example 3 The solvent for dissolving 2,6-dimethylnaphthalene was changed to various solvents such as heptane, cyclooctane, isooctane, octane, hexyl ether, nonane and decane, and 1 wt% of 2,6-dimethylnaphthalene was added thereto.
% Of the solvent, and 4 ml of each of these solvents was placed in a test tube having an inner diameter of 21 mm containing 4 ml of the medium shown in Table 2 and sterilized at 121 ° C. for 20 minutes. After cooling at room temperature, 1 platinum loop of Sphingomonas pouchimobilis A-7 strain was planted, and the test tube shaker was used at 30 ° C. and 300 rpm.
The cells were subjected to reciprocal shaking culture for several days. After culturing, the supernatant of the aqueous portion of this culture broth was treated with strong alkali and then analyzed by neutral high performance liquid chromatography (HPLC). As a result, it was found that the strain of the present invention can grow even when using various organic solvents and has sufficient conversion ability to 2,6-dimethylnaphthalene.

[0036]

Example 4 To a test tube having an inner diameter of 21 mm, 40 mg of 2,6-dimethylnaphthalene and the medium shown in Table 2 with a decalin concentration of 10 to 90% were added to bring the total amount to 10 ml, Sterilized at 121 ° C. for 20 minutes. After cooling at room temperature, Sphingomonas paucimobilis A-
7 strains were planted with 1 platinum loop, and 30 with a test tube shaker
Reciprocal shaking culture was performed at 300 ° C. for several days at 300 ° C. After culturing, the supernatant of the aqueous portion of this culture broth was treated with a strong alkali and then neutralized by high performance liquid chromatography (HPL
Analysis was performed in C), and the results shown in FIG. 1 were obtained. It was found that the strain of the present invention can grow even with various concentrations of decalin, and has sufficient conversion ability for 2,6-dimethylnaphthalene.

[0037]

Example 5 Two 500 ml Erlenmeyer flasks with baffles were each charged with 100 L medium (10 g / l bactotryptone, 5 g / l yeast extract, 5 g / l NaCl).
Then, 100 ml of 2,6-dimethylnaphthalene was added and sterilized at 121 ° C. for 20 minutes. After cooling at room temperature, 1 platinum loop of Sphingomonas pouchimobilis A-7 strain was inoculated using platinum loops and cultured at 30 ° C. for 5 days with shaking. After collecting the cells by centrifugation of the culture solution after culturing, in order to enhance the buffering ability of the medium and suppress the decrease in pH, among the medium components shown in Table 2, KH ₂ PO ₄ was added to (NH ₄ ) ₂ HPO. Instead of ₄ , washed with a medium (pH 7) containing 20 g / l and no NH ₄ NO ₃ added, suspend the cells in 4 ml of the same medium, and sterilize the test tube with an inner diameter of 21 mm Moved to
Further, 4 ml of decalin in which dimethylnaphthalene was dissolved in 1 wt% was added, and the mixture was shaken at 30 ° C. for 3 days to be reacted.
When the reaction product solution was analyzed by HPLC, the yield of 2,6-naphthalenedicarboxylic acid was 310 mg / l.

[0038]

According to the present invention, it is possible to grow in an organic solvent system comprising an organic solvent in which 2,6-dimethylnaphthalene is dissolved as a single carbon source or a conversion substrate and a medium containing no carbon source, and Efficiently producing 2,6-naphthalenedicarboxylic acid from 2,6-dimethylnaphthalene by using a producing bacterium capable of producing 2,6-naphthalenedicarboxylic acid, or a resting bacterium thereof, a treated product thereof or an enzyme derived from the bacterium. Can be produced well.

[Brief description of drawings]

FIG. 1 is a diagram showing the influence of production of 2,6-naphthalenedicarboxylic acid in Example 4 with a decalin concentration of 10 to 90%.

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[Procedure amendment]

[Submission date] November 18, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0014

[Correction method] Change

[Correction content]

[0014]

[Table 1]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] O029

[Correction method] Change

[Correction content]

[0029]

[Table 2]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12R 1:01) (72) Inventor Ryuichirou Kurane 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki (72) Inventor Naohisa Kamimura 1-13-32 Namiki, Kawaguchi City, Saitama Prefecture No. 609, Evergreen Kawaguchi (72) Inventor Yutaka Sakai 2-Fujigaoka, Midori-ku, Yokohama-shi, Kanagawa Prefecture 36-66 Greenhill Fujigaoka CII-114 (72) Inventor Masafumi Tobita 2021-1 Totsuka-cho, Totsuka-ku, Yokohama-shi, Kanagawa Hoyu Palace Totsuka 711 (72) Inventor Tomoyuki Makino 2-Bunkyo-cho, Zentsuji-shi, Kagawa 5-10 Ministry of Agriculture, Forestry and Fisheries Ikuno Camp 2-1

Claims (6)

[Claims] 1. A 2,6-naphthalenedicarboxylic acid-producing bacterium belonging to the genus Sphingomonas. 2. A 2,6-naphthalenedicarboxylic acid-producing bacterium belonging to the genus Sphingomonas and capable of producing 2,6-naphthalenedicarboxylic acid from 2,6-dimethylnaphthalene. 3. A 2,6-naphthalenedicarboxylic acid-producing bacterium which belongs to the genus Sphingomonas and has an ability to oxidize both methyl groups of 2,6-dimethylnaphthalene in an organic solvent system. 4. A 2,6-naphthalenedicarboxylic acid-producing bacterium in which the bacterium according to any one of claims 1 to 3 is Sphingomonas poutimobilis A-7. 5. The producing bacterium according to claim 1, 2, 6
-Culturing in an organic solvent-based medium composed of a medium and an organic solvent in which dimethylnaphthalene is dissolved to produce 2,6-naphthalenedicarboxylic acid 2,6-
A method for producing naphthalenedicarboxylic acid or a salt thereof. 6. The producing bacterium according to claim 1 or 2,
-Cultured in a medium containing or not containing dimethylnaphthalene or an organic solvent in which 2,6-dimethylnaphthalene is dissolved, and an organic solvent-based medium comprising the medium, and after collecting the cells, the cells or resting cells, or treatment thereof To produce 2,6-naphthalenedicarboxylic acid by reacting it in an organic solvent system with an enzyme derived from a substance or bacterial cells, or to convert 2,6-dimethylnaphthalene to 2,6-naphthalenedicarboxylic acid. Characteristic 2,6
-A method for producing naphthalenedicarboxylic acid or a salt thereof.