JPS59166092A - Preparation of octylglutaric acid - Google Patents

Abstract

PURPOSE:To obtain octylglutaric acid useful as an inhibitor for biosynthesis of fatty acid, by cultivating a specific microorganism belonging to the genus Gongronella, forming and accumulating octylglutaric acid in a culture solution, collecting it. CONSTITUTION:A microorganism belonging to the genus Gongronella, capable of producing octylglutaric acid, is cultivated, octylglutaric acid is formed and accumulated in a culture solution, and it is collected. The cultivation of the microorganism is carried out by subculture in corn meal agar medium, malt agar medium, Czapek and Dox agar medium, potato dextrose, etc., a grown mycelium in the medium is directly inoculated into a preparation medium in order to prepare octylglutaric acid, it is cultivated, and octylglutaric acid is formed and accumulated. Any of solid or liquid medium is used as a carbon source for the preparation medium, and the liquid medium is more preferably used.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing octylglutaric acid by fermentation.

As the microorganism used in the present invention, Gong I Nerabutleri 1i'l 3/K is used for Hyoguchi fishing.
O (Gongronella butleri M
3/gO) (FERM-Pt, tqs).

Its Dutch characteristics are as follows.

I Agar j@Ground Properties/) Growth on malt extract agar medium: Growth is moderate, and the colony diameter reaches 5 cTL in one week. The shape of the colony is fluffy.

The color is white.

Growth on potato agar medium: Properties similar to those on malt extract medium.

However, later it had a partition wall.

Chlamydospores are formed. Chlamydospores are usually hemiform, nuchal or internodal, spherical to hematocyst, pachycephalic, unbranched, or pseudoaxial to irregularly branched.

It wanders around in a distinct septum slightly below the sporangium.

The spores are spherical to hematocytoid in shape, 73 to 33 μm in diameter, and the membrane of the sporangium is smooth and dissolves to release a large number of spores. Pillar 714
i+ is hemispherical to dome shaped with a clear color, diameter da~/, 2μm0 with a clear apophysis at the bottom of the sporangium, apophysis is spherical to oblique, 3.3 to 33
μm0 Spores are colorless, smooth, oval to oval, often with flat sides, or kidney-shaped, square, λ-ri, 5×mu-o-yu0
gμ771. .

Zygospores are formed above the surface of the medium, H-type mating,
The zygospore-supporting stalk has no accessory branches, is homomorphic to slightly heteromorphic, and the zygospore is spherical, with a diameter of 77 to 3 g μm, brown to dark brown, and numerous wart-like protrusions. Heterotaritsuk.

Kawa Physiological properties Growth range pH: g-g, temperature/3-/IO℃ Genus level identification: This wild strain has /) polysporic sporangia, and the lower part of the sporangium. It was found that the fungus 3) belongs to the genus Gongronella of the family Mucoridae, which is a fungus of the order Mucorales, because it has a clear aphophysis and 3) has bulges and rhizoids.

Species identification: The culture properties and morphological characteristics of this wild strain were determined by Heraseltine & Ellie (J.
)'s Mycologia (Mycologia) k A(/
Gongronella batleri (9tlA) described in
The results showed good agreement with the properties of Gongronolla butleri).

Therefore, this wild strain was identified as Gongronella batlerii.

In addition, mating type G of Gongronella batleri, buttle
ri (+) stock (ENGFOgOgO) and G, butle
An attempt was made to crossbreed the ri(-) strain (TEFOgOg/) with this wild strain.
Zygospores were formed by crossing with the i(-) strain. Therefore, the wild strain used in the present invention is G, but]-eri(+
) was found to be a stock.

In the present invention, the microorganism can be cultured by a conventional method.

For example, it can be subcultured and accumulated on a cornmeal agar medium, a malt soybean medium, a Czapek Dox agar medium, or a potato dextrose agar medium.

The production medium may be either solid or liquid, but a liquid medium is more preferably used.

Carbon sources for the production medium include, for example, glucose, soluble carboxylic acid, glycerin, dextrin, sucrose, starch syrup, lactose, lactose, and maltose. be.

Nitrogen sources include peptone, meat extract, malt extract, yeast powder, soybean flour, peanut flour, cottonseed waste, cornstarch liquor, rice bran, and inorganic hydrangeas; , polypeptone, etc. are preferred.

Inorganic salts, such as calcium carbonate, magnesium sulfate,
By further adding potassium phosphate, sodium chloride, etc., growth of bacteria can be aided and production of the target product can be promoted.

Cultivation is carried out aerobically, and the pH is usually t −9
The temperature is selected from about 10 to θ°C, and the time is selected from about 1 to 300 hours.

The generated and accumulated octylglutaric acid can be extracted and purified by a known method.

For example, after extraction with ether, ethyl acetate, chloroform, etc. under acidic conditions, extraction with aqueous sodium bicarbonate, rinsing, extraction with the above solvent under acidic conditions, drying, and then purification by silica gel chromatography, etc. It is possible to obtain the target object.

The resulting octylglutaric acid or its pharmaceutically acceptable salts (eg, sodium salt, calcium salt, potassium salt, etc.) are useful as fatty acid biosynthesis inhibitors.

Next, the BA of the present invention will be explained in more detail with reference to examples.

Example/ Soluble starch/, 0%, Glucose J, 5%, Meat extract 0. ! ;%, soy protein λ, θ ratio, polypeptone O, S
%, Na01O, 3%, KH, Po, 0.05%, M
, 9SO, ・riH, Oθ, os, antifoaming agent (“CBII
Lycium ``''') O0θ/Tg (pHA; tg) was cultured for 30 days at C.3, and the seeds were added to a medium/S of the same composition at 30C and 2 g Orpm.
, cultured in 2J VVM.

The production amount of α-n-octylglutaric acid reached its peak on the 3rd day after the start of the culture, and the culture was discontinued on the 6th day.The bacterial cells were separated using a piece of paper, and the obtained culture fluid F/θl was added to concentrated hydrochloric acid. pH at
3.0, extracted once with ethyl acetate, and condensed with ethyl acetate to make 10 ml. S% baking soda water/'0
After extraction 5 times at 0 mA, acidify again with daughter hydrochloric acid and /co
The extract was extracted with 5 ml of ethyl acetate, nipped with Glauber's salt, and then concentrated to dryness under reduced pressure to obtain 9 g of both λ/λ and 9 g. Dry matter/, 2. l
le? "Wako" silica gel O-, 200 5lI-o
A crude fraction of α-n-octylglutaric acid eluted with benzene/jJ, then benzene: methylene chloride (/:/)/θl, and then methylene chloride: ethyl acetate (g:λ)/l on a column using P. 7 g of sulfur was obtained.

Furthermore, 57 g of silica kel Q −
, 200 and developed with benzene, /11 benzene:methylene chloride (/:/, 1.2., +7 methylene chloride-,27) to obtain α-n-octylglutaric acid as a pale yellow oil. .

The target identification can be performed using elemental analysis, mass spectrometry, "C-NM
R, 工Rsuhe // l-/lz, ``O-NMR,'H
- I read NMR.

Molecular weight, 2g1 Molecular formula 〇, a & 40n δ# from plate'O-NMR, 2. ．． 2 ppm s,
/7? From jppm s and 'H-NMR, δ//, J,
2ppm br, 2H, has two carboxyl groups from the engineering R spectrum, and +10-NMBδ/g, /pp
mQ, δ41.11. ．． 7 ppm d shows that it is a saturated dicarboxylic acid having 7 methylene groups and 7 methine moieties. Furthermore, from 'H-NMR, δ/, 9.2
ppm m, 2H is a derivative of glutaric acid because the 7 methylene groups are between the methine and methylene groups bonded to the carboxyl group, and C7I (,,
is an n-heptyl group according to NMR etc., so α
- It is thought that it can be absorbed by n-octylglutaric acid.

Furthermore, this result agreed with various instrumental analysis data of synthetic α-n-octylglutaric acid.

Reference example/ (Inhibitory activity of acetyl-coA carboxylase by α-n-octylglutaric acid) Numara no Kata (Mebo Z cup, (ji, 爲〃hair π1a 771 Tei, 7) ,2
7 years] Acetyl OOA carboxylase obtained from partial shoots obtained from D. 0.2 m Upit f, 3'OmM h
Lys-hydrochloric acid buffer (p) (,wa!;) (10mM citric acid, / On MM, 9OA+z, 3.7k
1.mM glutathione, containing 0.73 m9/ml bovine serum albumin) at 37°C. ? At the time of activation for θ minutes, Honokishin was added, and the fixation of H14CO,'- to malonyl OoA was measured. By adding SS μi/ml of this substance, inhibition of the activity was observed for about 2 hours.

Applicant Mitsubishi Chemical Industries Co., Ltd. Agent Patent Attorney Hase - Others / Famous Procedural Official Book (Method) July 9, 1980, Director General of the Office of the Attorney General Kazuo Wakasugi 1 Display of the case Patent Application No. 38605 of 1988 2 Name of the invention Process for producing octylglutaric acid 3 Person making the amendment! 1 Which relationship Patent applicant (59G) Mitsubishi Chemical Industries, Ltd. 4th generation 1 person Address: 2-5-2'' Marunouchi, Chiyoda-ku, Tokyo 100 21. Mitsubishi Chemical Industries, Ltd. ((1 person)

Claims (1)

[Claims] (1) Gon (ronella
) A method for producing octylglutaric acid, which comprises culturing a microorganism that has the ability to produce octylglutaric acid, producing and accumulating octylglutaric acid in a culture solution, and collecting the same.