JPH07313176A - New microorganism and production of 2, 6-naphthalene dicarboxylic acid using the same - Google Patents

Abstract

PURPOSE:To obtain a new microorganism belonging to the genus Fusarium or Aspergillus and capable of producing 2,6-naphthalene dicarboxylic acid useful as a raw material for highly functional resins, liquid crystals, medicines, coating pigments, etc., from 2,6-dimethylnaphthalene. CONSTITUTION:This new 2,6-naphthalene dicarboxylic acid-producing fungus belongs to the genus Fusarium [e.g. Fusarium solani YIII 5-2 strain (FERM P-14295)] or the genus Aspergillus [e.g. Aspergillus flavus YIII 5-2 strain (FERM P-14296)], and can produce 2,6-naphthalene dicarboxylic acid from 2,6 dimethylnaphthalene. The fungus is cultured in an organic solvent-containing medium comprising a medium and an organic solvent containing the 2,6- dimethylnaphthalene, and the product is subsequently recovered from the cultured product. The 2,6-naphthalene dicarboxylic acid useful as a raw material for highly functional resins, liquid crystals, polyamide medicines, dyestuffs, pigments, etc., is thus profitably obtained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel microorganism belonging to the genus Fusarium or Aspergillus, and a method for producing 2,6-naphthalenedicarboxylic acid by oxidizing 2,6-dimethylnaphthalene using the microorganism.

[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. Especially, the usage as polyethylene naphthalate (PEN) resin is said to be 80 to 90%, and compared with the current polyethylene terephthalate (PET) resin, the heat resistance temperature is about 35 ° C, and the breaking strength is also about 25 ° C 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 the reaction can be performed at room temperature and atmospheric pressure, and the functional group is not transferred, so that a by-product is 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 is not oxidized. Was not detected. (EA BARNSLEY, APPLIED A
ND ENVIRONMENTAL MICROBIO
LOGY 54, 428-433, 1988). or,
Co-oxidation with sugars by Nocardia bacteria 2,
Report of confirmation of 6-naphthalenedicarboxylic acid (G.K.S.
KRYABIN, et al. , DOKL. AKAD.
NAUK. USSR 202,973-974,197
2), but the yield was low and the study was limited to qualitative research.

More recently, a method for producing 2,6-naphthalenedicarboxylic acid by fermentation using a bacterium belonging to the genus Pseudomonas (Japanese Patent Laid-Open No. 3-80091) has an oxidizing power for alkyl groups of alkylnaphthalene compounds. A method for producing naphthalenedicarboxylic acid using a microorganism (Japanese Patent Laid-Open No. 15365/1993) has been proposed.

[0006]

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 almost no by-products are formed.
However, in the production by ordinary microbial reaction, the culture conditions for the production are mainly water-based, and moreover, as a conversion substrate,
6-Dimethylnaphthalene is a so-called solid-liquid reaction system that is solid at room temperature and insoluble in water, so that the supply amount of the conversion substrate is limited and the supply amount is difficult to control. It can be said that there are still problems in terms of production. Furthermore, the water-based fermentation method is
The problem of foaming due to the action of microorganisms has always occurred, and it has always been a problem that the dissolved oxygen concentration is lowered and the aeration amount and stirring speed are limited. As a countermeasure,
So-called anti-foaming agents such as silicone and soybean oil are used, but the effect does not last long, and therefore, the process was unnecessarily complicated, and it sometimes became an obstacle to productivity improvement. .

In view of the problems of the method for producing 2,6-naphthalenedicarboxylic acid when the aqueous fermentation method based on the above-mentioned conventional technique is used, the object of the present invention is to obtain a heterogeneous system, that is, a large amount of organic solvent and aqueous liquid. By setting the reaction environment as follows and directly producing 2,6-naphthalenedicarboxylic acid as the target substance from 2,6-dimethylnaphthalene as the starting substance in the reaction system, the above-mentioned problems are overcome and production is performed. It is intended to provide a manufacturing method with good efficiency.

Therefore, a microorganism having an ability to oxidize both methyl groups of 2,6-dimethylnaphthalene, capable of growing or reacting in an organic solvent-mediated system, and not inhibiting the oxidation reaction was found, When 2,6-dimethylnaphthalene dissolved in an organic solvent is supplied to such a microorganism, it becomes possible to continuously convert a large amount into 2,6-naphthalenedicarboxylic acid by an organic solvent-mediated liquid-liquid reaction system. Clearly, it will be of great utility to the petroleum and petrochemical industries.

[0009]

Under these circumstances, the present inventors have conducted extensive studies in order to solve the above problems, developed an effective method for screening microorganisms, and carried out efficient search for microorganisms. As a result, we found a novel microorganism capable of efficiently oxidizing the methyl groups on both sides of 2,6-dimethylnaphthalene dissolved in an organic solvent.
The present invention has been accomplished by enabling a manufacturing method using a liquid reaction system.

That is, the present invention relates to the genus Fusarium (Fusa).
rium) or Aspergillus (Aspergillus)
A 2,6-naphthalenedicarboxylic acid-producing bacterium from 2,6-dimethylnaphthalene dissolved in an organic solvent is used as an organic solvent-mediated two-phase system (liquid-liquid system) comprising an organic solvent in which 2,6-dimethylnaphthalene is dissolved and an aqueous medium. The present invention provides a method for producing 2,6-naphthalenedicarboxylic acid, which comprises culturing or converting in a liquid system).

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

[0012] For example, for soil collected 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) is dispensed into a test tube and sterilized, and then the soil is sterilized. In addition, an organic solvent in which 2,6-dimethylnaphthalene or 2,6-dimethylnaphthalene is dissolved, for example, decalin, is added and cultivated, or from the viewpoint of screening efficiency, in order to suppress the growth of unnecessary bacteria, Chloramphenicol is added and culturing is performed by a test tube shaker or the like. This culture solution was subcultured in a similar medium previously dispensed into another test tube, and then 2,6-dimethylnaphthalene was added,
Further incubate with a test tube shaker. 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 colonies obtained by the culture were isolated, and 2,6-
By confirming the production capacity of naphthalenedicarboxylic acid, a 2,6-naphthalenedicarboxylic acid-producing microorganism can be selected.

As a medium for separating these microorganisms, general medium components other than carbon sources can be used. That is, with respect to 1 liter of distilled water or ion-exchanged water,
Examples of the nitrogen source include ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate, ammonium nitrate, and urea, and examples of the inorganic salts include potassium, sodium, calcium, iron, magnesium,
Various salts such as manganese and copper can be used. In addition, the above-mentioned culture conditions may generally be culture conditions in which microorganisms are not killed, and for example, the culture conditions are aerobically carried out at a pH of about 3-9 and a temperature of about 15-40 ° C.

The confirmation of the ability of the obtained microorganisms to produce 2,6-naphthalenedicarboxylic acid was carried out by culturing each of the microorganisms in a medium containing 2,6-dimethylnaphthalene as a carbon source under the same conditions as above. The supernatant of the prepared culture solution is subjected to an appropriate analytical method, for example, high performance liquid chromatography (H
PLC), gas chromatography (GC), nuclear magnetic resonance spectrum (NMR), infrared absorption spectrum (I
R), ultraviolet absorption spectrum (UV), etc. may be used for analysis. The obtained microorganism was tested for identification, and had the properties shown in Tables 1 and 2 below.

[0015]

[Table 1]

[0016]

[Table 2]

The present microorganism is a filamentous fungus which 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. A filamentous fungus producing 2,6-naphthalenedicarboxylic acid having such characteristics has not been reported yet.

From the above mycological properties, the present inventors
The II31-3 strain is Fusarium solan.
i), YII15-2 strain is Aspergillus flavus (A
spergillus flavus), a new strain belonging to
This bacterium was designated as Fusarium solani YII31-3 strain and Aspergillus flavus YII15-2 strain and deposited at the Institute of Biotechnology, Institute of Industrial Science (FERM).
P-14295, FERM P-14296).

The microorganism of the present invention also includes microorganisms belonging to Fusarium solani and Aspergillus flavus, which are capable of oxidizing the 2,6-methyl group, and natural and artificial mutants thereof.

Next, the method for producing 2,6-naphthalenedicarboxylic acid according to the present invention will be described with reference to Fusarium solani YII3.
1-3 (FERM P-14295) strain and Aspergillus flavus YII15-2 (FERM P-14
296) is used as an example.

As a medium component for culturing these filamentous fungi, other than the carbon source and 2,6-dimethylnaphthalene as a conversion substrate, other medium components may be used if they grow well. There is no particular limitation. That is, as a growth substrate, for example, ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium carbonate, ammonium acetate, ammonium nitrate, urea and the like as a nitrogen source, and inorganic salts such as potassium, sodium, iron, magnesium, manganese and copper. Various salts such as calcium, calcium and cobalt can be used. In addition to 2,6-dimethylnaphthalene as a carbon source, salicylic acid,
It is also possible to add 2-methylnaphthalene, anthracene, glucose, fructose, starch or the like as an auxiliary substrate. Also, in the case of filamentous fungi, the reactivity may greatly change depending on the type of nitrogen source of the growth substrate, so in order to obtain better reactivity, the type of nitrogen source should be selected according to the type of fungus. Is preferably selected.

As the solvent for dissolving 2,6-dimethylnaphthalene used in the organic solvent-mediated culture, for example, heptane, n-octane, isooctane, nonane, decane,
Dodecane, tetralin, hexyl ether, etc. can be used, but they do not decrease in volatilization in culture for several days, and the microbial toxicity is neither too strong nor too weak in order to suppress the growth of various bacteria, and 2 Solvents other than the above can also be used as long as they do not assimilate and dissolve 6,6-dimethylnaphthalene. And any one or more of these may be arbitrarily mixed and used.

The method of dissolving 2,6-dimethylnaphthalene in a solvent and feeding the same as in the present invention, compared to the solid-liquid reaction method of feeding 2,6-dimethylnaphthalene directly to a conventional 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, in the present bacterium, 2,6-naphthalenedicarboxylic acid can be produced by directly supplying 2,6-dimethylnaphthalene without dissolving it in a solvent as in the conventional method.

[0024] The culture conditions may be such that the present bacterium can be proliferated without being killed. For example, the culture temperature is about 10 to 40 ° C, more preferably about 20 to 35 ° C, and the pH of the medium is about 3 to 9, more preferably Is preferably in the range of 4 to 7 and aerobically cultured or reacted for about 1 to 30 days.

In order to efficiently produce the desired 2,6-naphthalenedicarboxylic acid by culturing these filamentous fungi using 2,6-dimethylnaphthalene as a single carbon source or conversion substrate, 2,6-dimethylnaphthalene is used. It is necessary to add naphthalene at least in an amount necessary for growing these filamentous fungi. 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, aqueous ammonia, potassium hydroxide, etc., or 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 obtained bacterial cell culture can be used as it is as an enzyme source, but it is preferable to use a microbial cell 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 bacterial cells can be purified by a conventional method, and when produced using these resting bacterial cells, treated bacterial cells or enzymes,
It can be performed under the culture conditions. The process of producing 2,6-naphthalenedicarboxylic acid from 2,6-dimethylnaphthalene using these filamentous fungi may be a batch process,
It is also possible to use 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. 2,6 in culture or reaction
-The naphthalenedicarboxylic acid may be purified by a conventional method. That is, 2,6-naphthalenedicarboxylic acid can be recovered by adding a culture solution or a reaction solution to acidify it. 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 has been made possible by finding a microorganism capable of efficiently oxidizing the methyl groups on both sides of 2,6-dimethylnaphthalene dissolved in an organic solvent.

[0028]

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

[0029]

Example 1 Separation of 2,6-dimethylnaphthalene-utilizing bacteria resistant to organic solvents

Chloramphenicol was added to 50 μg / ml in YM medium (yeast extract 3 g, peptone 5 g, malt extract 3 g, glucose 10 g, pH 5.
Put 5 ml of 5) into a test tube with an inner diameter of 21 mm, add 1 scoop of soil to the test tube from soil collected from all over the country, and use a test tube shaker at 30 ° C. and 270 rpm.
Shake culture overnight.

Next, among the components necessary for the growth of microorganisms, a medium containing no carbon source (BACTO YEASTNITROGENBASE (Difco
(Hereinafter, referred to as YNB)) was placed in a test tube having an inner diameter of 21 mm in an amount of 4 ml, and further contained an organic solvent in which 2,6-dimethylnaphthalene was dissolved, that is, 2,6-dimethylnaphthalene (1 (w / v)%). Add 4 ml decalin,
Sterilize at 121 ° C for 20 minutes.

After cooling, chloramphenicol was added to the culture medium of the test tube at 50 μg / ml, and the above YM was added.
400 μl of the culture medium is subcultured using a micropipette, and further shake-cultured by a test tube shaker.

To this culture solution, the same chloramphenicol was added again at a concentration of 50 μg / ml.
-Decalin containing dimethylnaphthalene and a medium are mixed and dispensed into another test tube, 40 μl of which is subcultured using a micropipette, and again shake-cultured by a test tube shaker.

This culture solution or a culture solution diluted with sterilized water or the like was applied to the same YNB agar medium, and 2,6-dimethylnaphthalene was directly supplied to further culture.

369 strains of bacteria that assimilated 2,6-dimethylnaphthalene and appeared on a plate medium were separated from 86 strains of fungi, and filamentous fungi were selected from these to isolate Fusarium solani YII31- 3 strains and Aspergillus flavus YII15-2 strain were obtained.

[0036]

Example 2 Production of 2,6-naphthalenedicarboxylic acid by an organic solvent-resistant assimilating bacterium

Of the components necessary for the growth of microorganisms, a nitrogen source-free medium (BACTO YEAST CORBONBASE (manufactured by Difco): hereinafter referred to as YCB) was added to ammonium nitrate 0.5.
Two types of media were prepared to have a concentration of mg / l or ammonium sulfate of 0.5 mg / l. 4 ml of each medium prepared by changing the type of nitrogen source was placed in a test tube with an inner diameter of 21 mm, and 1 part of 2,6-dimethylnaphthalene was added.
Add 4 ml of decalin containing (w / v)%, 121 ° C
It was sterilized for 20 minutes, and this was used as an organic solvent-mediated medium.
Separately, in order to compare the effect with organic solvent mediated culture,
Cultivation without an organic solvent was also performed at the same time. That is, 4 ml of a medium prepared by adding 0.5 mg / l of ammonium nitrate to a YCB medium was placed in a test tube, and diethyl ether containing 5 (w / v)% 2,6-dimethylnaphthalene was sterilized by a filter. 0.8 ml was added, and the mixture was allowed to stand to evaporate diethyl ether to precipitate crystalline 2,6-dimethylnaphthalene, which was used as an organic solvent-free medium (solid-liquid system).

The YII31-3 strain and the YII15-2 strain isolated in Example 1 were tested for growth and production in the above-mentioned three types of media. That is, from the culture solution of the YM medium in which the bacterial cells have been cultured in advance, 40 cells are added to each of the above three types of medium.
μl was inoculated and cultured with shaking at 30 ° C. and 270 rpm.
The results are shown in Table 3 below.

[0039]

[Table 3]

From the results shown in Table 3 above, these two bacterial strains of the present invention have the ability to produce 2,6-naphthalenedicarboxylic acid from 2,6-dimethylnaphthalene in an organic solvent-mediated two-phase system. It is clear that the filamentous fungus of the genus Fusarium has a higher productivity in the organic solvent-mediated two-phase system than the conventional solid-liquid system, and is a more suitable bacterial species for the two-phase system culture. Was also confirmed.

[0041]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to grow in an organic solvent-mediated culture system of an organic solvent in which 2,6-dimethylnaphthalene is dissolved as the sole carbon source or conversion substrate, and a medium containing no carbon source. Alternatively, 2,6-naphthalenedicarboxylic acid can be produced in an organic solvent-mediated culture system of an organic solvent in which 2,6-dimethylnaphthalene is dissolved as the sole carbon source or conversion substrate and a medium containing the carbon source. It is possible to efficiently produce 2,6-naphthalenedicarboxylic acid from 2,6-dimethylnaphthalene using a producing bacterium, or a resting bacterium or an enzyme derived from the bacterium.

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Claims (4)

[Claims] 1. A 2,6-naphthalenedicarboxylic acid-producing bacterium belonging to the genus Fusarium or Aspergillus and capable of producing 2,6-naphthalenedicarboxylic acid from 2,6-dimethylnaphthalene. 2. A 2,6-naphthalenedicarboxylic acid-producing bacterium which belongs to the genus Fusarium or the genus Aspergillus and has an ability to oxidize both methyl groups of 2,6-dimethylnaphthalene in an organic solvent-mediated system. 3. A 2,6-naphthalenedicarboxylic acid producing bacterium, wherein the producing bacterium according to claim 1 or 2 is Fusarium solani YII31-3 or Aspergillus flavus YII15-2. 4. The production strain according to claim 1 is 2,6
-Culturing in an organic solvent-mediated medium consisting of a medium and an organic solvent in which dimethylnaphthalene is dissolved to produce 2,6-naphthalenedicarboxylic acid 2,
A method for producing 6-naphthalenedicarboxylic acid or a salt thereof.