JP2715260B2 - New green algae, new yeast, and method for producing 2,6-naphthalenedicarboxylic acid using the green algae or the yeast - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing 2,6-naphthalenedicarboxylic acid by oxidizing 2,6-dimethylnaphthalene using a green algae belonging to the genus Protoceca or a yeast belonging to the genus Candida and the organism. .

[0002]

2. Description of the Related Art 2,6-Naphthalenedicarboxylic acid or an ester derivative thereof is a compound useful as a raw material for highly functional resins, a raw material for liquid crystals, a raw material for polyamide-based pharmaceuticals, and dyes and pigments. Yes, it is produced. In particular, the use as polyethylene naphthalate (PEN) resin is said to be 80 to 90%, compared to the current polyethylene terephthalate (PET) resin.
It is expected to be mass-produced as having high performance of about 35 ° C. in heat-resistant temperature and about 25 ° C. in breaking strength, and excellent in secondary 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 the transfer of a functional group easily occurs, it is difficult to obtain 2,6-naphthalenedicarboxylic acid of high purity, and the high-temperature and high-pressure conditions are not suitable. It requires energy, consumes a large amount of energy, and has problems such as environmental pollution.

[0003]

As a method for solving these problems, research on a microorganism oxidizing 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 carried out at normal temperature and normal pressure and the transfer of a functional group does not occur, so that almost no by-product is generated.

However, it has been reported that even when 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, VOL. 54,428-433,198
8). In addition, a report of confirmation of 2,6-naphthalenedicarboxylic acid by co-oxidation with carbohydrates by bacteria of the genus Nocardia (GK SKRYABIN, et al., DOK)
L. AKAD. NAUK. USSR 202,973 ~
974, 1972), but the yield was low and it was a qualitative study.

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

[0006] The oxidation method using a microorganism as a catalyst has excellent characteristics that it can be reacted at normal temperature and normal pressure and that there is almost no by-product.

[0007] Therefore, if a bioreactor capable of oxidizing 2,6-dimethylnaphthalene at both methyl group terminals and capable of converting it to 2,6-naphthalenedicarboxylic acid in a large amount can be constructed, energy-saving and environmentally friendly chemistry can be achieved. As a product manufacturing process, the petroleum and petrochemical industries have great utility.

[0008]

Under these circumstances, the present inventors have conducted intensive studies to solve the above-mentioned problems and conducted an efficient search for effective organisms. A novel organism capable of efficiently oxidizing methyl groups on both sides has been found, a production method by production has been made possible, and the present invention has been accomplished.

That is, the present invention relates to the genus Prototheca (Pro
An object of the present invention is to provide a method for producing 2,6-naphthalenedicarboxylic acid from 2,6-dimethylnaphthalene using a green algae belonging to C. totheca) or a yeast belonging to the genus Candida.

The novel green algae or novel yeast of the present invention is grown on a medium to which only 2,6-dimethylnaphthalene is added as a carbon source, and is screened for its ability to produce 2,6-naphthalenedicarboxylic acid. Can be separated by

[0011] Among the soil collected from all over the country, of the components necessary for the growth of these organisms, a medium containing no carbon source (hereinafter referred to as a medium) is dispensed into test tubes, sterilized and added to the soil. Then, 2,6-dimethylnaphthalene is further added, and the cells are cultured using a test tube shaker or the like. After inoculating this culture solution into the same medium previously dispensed into another test tube, 2,6-dimethylnaphthalene is added, and further cultured by a test tube shaker or the like. The culture solution after culturing is diluted with the above-mentioned medium or sterilized water, applied to an agar medium or the like containing no carbon source, and then further cultured by supplying 2,6-dimethylnaphthalene. The colonies obtained by the culture were isolated, and for each strain, 2,
By confirming the production capacity of 6-naphthalenedicarboxylic acid, it is possible to select a fine production of 2,6-naphthalenedicarboxylic acid.

As the medium for separating these organisms, general medium components other than the carbon source can be used. That is, for 1 liter of distilled water or ion-exchanged water, for example, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate, ammonium nitrate, urea, etc., and inorganic salts, for example, potassium, sodium, calcium, iron , Magnesium, manganese, copper, and other salts can be used. In addition, the culture condition may be any culture condition that generally does not kill the organism, and is, for example, aerobically performed at a pH of about 3 to 9 and a temperature of about 15 to 14 ° C.

To confirm the ability of the obtained organisms to produce 2,6-naphthalenedicarboxylic acid, each organism was cultured in a medium containing 2,6-dimethylnaphthalene as a carbon source under the same conditions as described above. The supernatant of the culture solution is analyzed by a suitable analytical method, for example, high performance liquid chromatography (HPLC), gas chromatography (GC), nuclear magnetic resonance spectrum (N
A desired product can be obtained by analyzing 2,6-naphthalenedicarboxylic acid using MR), infrared absorption spectrum (IR), ultraviolet absorption spectrum (UV) and the like.
In the present invention, the YII-20-1 strain and Y
II-21 strain was isolated and obtained.

In order to identify these organisms, various literatures (Keisuke Tsubaki: Fungi, 29, 155 (198)
8), Paula Arnold and D.A. G. FIG.
Aearn: Mycology, 64, 265 (1
972); B. Cooke: The Journal
al of the Mitchell Society
y, 84, 213 (1968); B. Cook
e: The Journal of the Mitch
cell, 84, 217 (1968),
K. Tubaki and M.S. Soneda: Na
Gaoa, 6, 25 (1959); J. W. Kr
eger-van Rij: "The Yeasts"
(1984) Elsevier Science Pu
blishers B. V. The test was performed with reference to Table 1. As a result, the test piece had properties shown in Tables 1 and 2.

[0015]

[Table 1]

[0016]

[Table 2]

From the above biological properties, the present inventors suggest that Y
The II-20-1 strain was transformed from the green alga Protoceca genus, YII-21
The strain was identified as an organism belonging to the genus Candida. Further searching according to the literature, the YII-20-1 strain
It was considered Prototheca stagnora, but it was recognized that the assimilation of n-propanol was recognized.
The possibility of rototheca zopfii was also considered and the species was not determined. Table 3 shows the results of comparison with the same strains.
Shown in YII-21 strain is Candidapara.
The strain was determined to be a new strain of psilosis. Therefore, the present micro-organisms are each made of Prototheca sp. (Pr
othetheca sp. ) YII-20-1 strain and Candida parapsilosis
apsilosis) strain YII-21, of which Candida parapsilosis YII-21 strain was deposited at the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology [FE]
RM P-14382]. In addition, Protoseca sp. YI
Deposit was refused because the I-20-1 strain is a green algae.

[0018]

[Table 3]

These organisms can grow using 2,6-dimethylnaphthalene as a single carbon source and have 2,6-dimethylnaphthalene in a medium.
Accumulates 6-naphthalenedicarboxylic acid. Green algae and yeast producing 2,6-naphthalenedicarboxylic acid having such properties have not yet been reported.

The microscopic organism of the present invention comprises Prototheca s
p. , And belonging to Candida parapsilosis,
The present invention also includes microscopic organisms capable of oxidizing the methyl group at the 2,6-position and natural and artificial mutant organisms thereof.

Next, the method for producing 2,6-naphthalenedicarboxylic acid according to the present invention is described in Prototheca sp. YII-
20-1 strain or Candida parapsilosis YI
The case where the I-21 strain is used will be described as an example.

When cultivating the green algae or yeast of the present invention, the medium components include a carbon source and a conversion substrate of 2,6.
Except for containing dimethylnaphthalene, other medium components are not particularly limited as long as the growth of the microscopic organism is good. That is, in a nitrogen source as a growth substrate, for example, ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium carbonate, ammonium acetate, ammonium nitrate, urea, etc., and as inorganic salts, for example, potassium, sodium, iron, magnesium, manganese,
Salts such as copper, calcium, and cobalt can be used.
As a carbon source, salicylic acid, 2-methylnaphthalene, anthracene, glucose, fructose, starch and the like can be added as an auxiliary substrate in addition to 2,6-dimethylnaphthalene.

The culture conditions may be such that the microorganisms can be proliferated without dying. 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, it is aerobically cultured or reacted in the range of 4 to 7 for about 1 to 30 days.

In order to efficiently produce the desired 2,6-naphthalenedicarboxylic acid by culturing these green algae or yeast using 2,6-dimethylnaphthalene as a single carbon source or a conversion substrate, 2,6-dimethylnaphthalene is required. It is necessary to add dimethylnaphthalene at least in an amount necessary for green alga or yeast to grow. This microscopic organism is
Since it is hardly affected by 2,6-dimethylnaphthalene, it can be added in a large amount.
When the amount of 2,6-naphthalenedicarboxylic acid generated increases, the pH of the medium decreases, so that an alkaline solution such as sodium hydroxide, aqueous ammonia, potassium hydroxide or the like is supplied, or a component having a buffer capacity is added. If the pH is adjusted to an appropriate range by supplying or using the medium in advance, more efficient production can be achieved. Further, after the biological cells are cultured and grown in advance under the above conditions and collected, they may be subjected to the method described below.

Although the obtained cultured microbial cells can be used as an enzyme source as they are, it is preferable to use microbial cells obtained by solid-liquid separation by an operation such as centrifugation. Furthermore, the microscopic biological cells can be washed with a solution such as a phosphate buffer and suspended in the solution before use. In addition, enzymes derived from microscopic living cells can be purified by a conventional method, and when they are produced using these resting cells, treated cells or enzymes, they can be carried out under the culture conditions described above. The step of producing 2,6-naphthalenedicarboxylic acid from 2,6-dimethylnaphthalene using these green algae or yeast may be a batch type or a bioreactor. Further, the microscopic biological cells, the processed product thereof, or the enzyme can be immobilized according to a usual immobilization method using, for example, a calcium alginate method, a polyacrylamide method, a polyurethane resin method, a photocrosslinkable resin method, or the like. 2,6-Naphthalenedicarboxylic acid in the culture solution or the reaction solution may be purified according to a conventional method. That is, 2,6-naphthalenedicarboxylic acid can be recovered by adding a culture solution or a reaction solution and acidifying the mixture. Also,
It can be recovered 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 is possible by finding green algae and yeast which can utilize 2,6-dimethylnaphthalene and can efficiently oxidize methyl groups on both sides of 2,6-dimethylnaphthalene. It has become.

[0027]

EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to the following examples.

[0028]

Example 1 Among the components required for the growth of microscopic organisms, a medium containing no nitrogen source (BACTO YEAST CORBO)
NBASE (manufactured by Difco)) and ammonium nitrate 5
A medium added to a concentration of 0.2 mg / l was prepared,
8 ml was placed in a 1 mm test tube and sterilized at 121 ° C. for 20 minutes. Then, 5% filter-sterilized 2%
0.8 ml of diethyl ether containing 6-dimethylnaphthalene was added, and the diethyl ether was allowed to evaporate to precipitate crystalline 2,6-dimethylnaphthalene.

The isolated Prototheca sp. YII-20
-1 strain and Candida parapsilosis YII-
For 21 strains, growth production was tested on the above medium in which 2,6-dimethylnaphthalene was used as the sole carbon source and crystalline 2,6-dimethylnaphthalene was precipitated. That is, YM (3 g of yeast extract, 5 g of peptone, 3 g of malt extract, 10 g of glucose, pH5.
5) 80 μl of the culture solution of the culture medium was inoculated into a culture medium for growth and production, and cultured with shaking at 30 ° C. and 270 rpm for 14 days.
The results shown in Table 4 were obtained.

[0030]

[Table 4]

From the results shown in Table 4, it was confirmed that these green algae and yeast had an ability to produce 2,6-naphthalenedicarboxylic acid from 2,6-dimethylnaphthalene.

[0032]

According to the present invention, in a medium which can grow using only 2,6-dimethylnaphthalene as a carbon source and contains 2,6-dimethylnaphthalene as a conversion substrate,
Efficient production of 2,6-naphthalenedicarboxylic acid from 2,6-dimethylnaphthalene using production micro-organisms capable of producing 2,6-naphthalenedicarboxylic acid, or cells derived from the micro-organisms thereof or cells-derived enzymes. be able to.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication (C12N 1/16 C12R 1:72) (C12P 7/44 C12R 1:89) (C12P 7/44 C12R 1:72)

Claims (4)

(57) [Claims] 1. A green algae belonging to the genus Prototheca, comprising 2,6
-Prototheca sp. Capable of producing 2,6-naphthalenedicarboxylic acid from dimethylnaphthalene sp. YII
-20-1. 2. Candida parapsilosis Y belonging to the genus Candida and capable of producing 2,6-naphthalenedicarboxylic acid from 2,6-dimethylnaphthalene.
II-21. 3. The method according to claim 1, wherein said prototheca sp. YII-20-1 or Candida parapsilosis YII-21 is represented by 2,
Cultured in a medium containing 6-dimethylnaphthalene,
A method for producing 2,6-naphthalenedicarboxylic acid or a salt thereof, which comprises producing 2,6-naphthalenedicarboxylic acid. 4. The method according to claim 1, wherein said prototheca sp. YII-20-1 or Candida parapsilosis YII-21 is represented by 2,
Culturing 6-dimethylnaphthalene in a medium containing 2,6-dimethylnaphthalene, and producing 2,6-naphthalenedicarboxylic acid using the obtained culture, algae, fungi, or a treated product thereof; Characteristic 2,6-
A method for producing naphthalenedicarboxylic acid or a salt thereof.