JPH0371117B2 - - Google Patents

Description

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

Specifically, the microorganism used in the present invention is Gongronella batleri 3180 (Gongronella
butleri M 3180) (FERM-P6975).

Its mycological properties are as follows.

Properties on agar medium 1. Growth on malt extract agar medium: Growth is moderate, with colonies reaching 5 cm in diameter in one week. The colony is fluffy in shape. The color is white.

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

Microscopic properties The vegetative hyphae are branched, initially lacking septa but later gaining septa.

Forms stolons. The stolons have rhizoids, but the development of rhizoids is not very good. Chlamydospores are formed in the vegetative hyphae. Chlamydospores are usually solitary, terminal or internodal, spherical to subglobose, thickened, 3.5-9.2 μm.
Sporangial stalks arise from stolons and vegetative hyphae, are 2-7 μm wide, colorless, unbranched, or pseudoaxial to irregularly branched. There is a clear septum slightly below the sporangium.

The sporangium is spherical to subspherical, 15 to 35 μm in diameter, the membrane of the sporangium is smooth, and it dissolves to release many spores. The axis of the column is hemispherical to dome-shaped, with a distinct color, and a diameter of 4 to 12 μm. There is a clear apophysis at the bottom of the sporangium, which is hemispherical to bell-shaped, 3.3 to 33 μm.

Spores colorless, smooth, oval to oval, often flattened on one side, or kidney-shaped, 2.2−4.5×1.6
−2.8μm.

Zygospores are formed above the surface of the medium, H-type mating, the zygospore-supporting stalk has no accessory branches,
Almost homomorphic to slightly heteromorphic, zygospores subglobose, diameter 17
~38μm, ~dark and colored, with numerous wart-like protrusions. Heterotaritsuk.

Physiological properties Growth range PH: 4-8, temperature 15-40℃ Identification at the genus level: This wild strain 1) has polysporangial sporangia, 2) clear apophysis at the bottom of the sporangium. 3) It was found to belong to the genus Gongronella of the family Mucoridae, which is a fungus of the order Mucorales, because it has bulges and rhizoids.

Species identification: The culture properties and morphological characteristics of this wild strain were described by Hesseltine & Ellis.
Gongronella batleri (Gongronella) described in Mycologia 56 (1964)
butleri).

Therefore, the present wild strain is Gongronella batlerii.
was identified.

In addition, the mating type G of Gongronella batleri.
Butleri (+) strain (IF08080) and G. butleri (-) strain (IFO08081) were tried to cross with this wild strain. formed spores. Therefore, the wild strain used in the present invention was found to be the G. butleri (+) strain.

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

For example, it is subcultured on cornmeal agar, malt agar, zebra agar, potato dextrose agar, etc., and the growing mycelium of the medium is directly inoculated into the production medium for octylglutaric acid production. It can be cultured, produced and accumulated.

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

Examples of the carbon source of the production medium include glucose, soluble starch, glycerin, dextrin, sucrose, starch syrup, molasses, lactose, and maltose, with glucose and soluble starch being preferred.

Nitrogen sources include peptone, meat extract, malt extract, yeast extract, soybean flour, peanut flour, cottonseed meal, cornstarch liquor, rice bran, inorganic nitrogen sources, etc. Meat extract, soybean flour, polypeptone, etc. is preferred.

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

Cultivation is carried out aerobically, and the pH is usually 5 to 5.
9. The temperature is selected from about 20 to 40°C, and the time is selected from about 24 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, extraction again with the above solvent under acidic conditions, concentration to dryness, and then purification by silica gel chromatography, etc. It is possible to obtain the desired 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 present invention will be explained in further detail with reference to Examples.

Example 1 Soluble starch 1.0%, glucose 3.5%, meat extract 0.5%, soy protein 2.0%, polypeptone 0.5%,
NaC2.5%, _KH2PO40.05 %, _{MgSO4 ・ 7H2}
O 0.05%, antifoaming agent (“CB442”) 0.01% (PH5.8)
Using 3% of the seeds cultured at 30°C for 2 days in a medium containing
Cultured at 280 rpm and 2.3 VVM.

The amount of α-n-octylglutaric acid produced reached its peak on the 3rd day after the start of the culture, and on the 5th day, the culture was discontinued, the bacterial cells were separated using paper, and the resulting culture solution was
10 was adjusted to pH 3.0 with concentrated hydrochloric acid, extracted twice with ethyl acetate, and the ethyl acetate layer was concentrated to 100 ml. After extracting 5 times with 100 ml of 5% sodium bicarbonate solution, acidify again with concentrated hydrochloric acid, extracting 5 times with 100 ml of ethyl acetate,
After drying with Glauber's salt, the residue was concentrated to dryness under reduced pressure to obtain 12.9 g of fractions. 12.4g of dry matter was added to “Wako” silica gel C-200.
A column using 240 g of 2.7 g of a crude fraction of α-n-octylglutaric acid was eluted with 1 part of benzene, then 10 parts of benzene:methylene chloride (1:1), and then 1 part of methylene chloride: ethyl acetate (8:2). Obtained.

Furthermore, this fraction was added to a column using 54 g of silica gel C-200 to prepare benzene 2.1, benzene:methylene chloride (1:1), and 2.2 methylene chloride.
2.2 to obtain α-n-octylglutaric acid as a pale yellow oil.

The target identification can be performed using elemental analysis, mass spectrometry, ¹³
C-NMR, IR spectrum, ^13C -NMR, ^1H-
I used NMR.

Molecular weight 244 Molecular formula C ₁₃ H ₂₄ O ₄ ¹³ From C-NMR δ182.2ppms, 179.6ppms and ¹
According to H-NMR, it has δ11.22ppm br2H, and according to IR spectrum, it has two carboxyl groups, and ¹³ C-
NMR δ14.1ppmQ and δ44.7ppmd indicate that it is a saturated dicarboxylic acid having one methylene group and one methylene moiety. Furthermore, from ^1H -NMR, δ1.92ppm m 2H has one methylene group, or it is a derivative of glutaric acid because it is between the methylene group and the methylene group bonded to the carboxyl group, and C ₇ H ₁₅ is bonded to the α position. is considered to be α-n-octylglutaric acid since it is an n-heptyl group according to NMR etc.

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

Reference example 1 (α-n- of acetyl-CoA carboxylase
Inhibitory activity by octylglutaric acid) Numa et al.'s method for determining the fixation of H ¹⁴ CO ^- ₃ to malonyl-CoA (Methods in Enzymology 71, p. 5, 1981)
0.2 m Unit of partially purified acetyl-CoA carboxylase obtained by
The substance was added at the time of activation for 30 minutes at 37° C. (containing C ₂ , 3.75 mM glutathione, and 0.75 mg/ml bovine serum albumin), and the fixation of H ¹⁴ CO ⁻ ₃ to malonyl-CoA was measured. Approximately 50% inhibition of activity was observed by adding 55 μg/ml of this substance.

Claims (1)

[Claims] 1. A method for producing octylglutaric acid, which comprises culturing a microorganism belonging to the genus Gongronella and having the ability to produce octylglutaric acid, producing and accumulating octylglutaric acid in a culture solution, and collecting the same.