JPH08196288A - Production of long-chain fatty acid - Google Patents

Abstract

PURPOSE: To efficiently obtain long-chain fatty acids which are frequently utilized as a starting substance for oleochemicals in industry by culturing a microorganism, Francisella sp. WYSA-40-2 and collecting the product from the culture mixture. CONSTITUTION: Francisella sp. strain: WYSA-40-2 (FERM P-14621) is inoculated to the culture medium, to which calcium carbonate or the like is added to keep the pH at 6-8 and shaking culture is continued at 20 deg.C for 2 days to produce and accumulate long-chain fatty acids in cell bodies. When the products reach the maximum in the culture mixture, the mixture is centrifuged to separate the cell bodies, which are freeze-dried, crushed, extracted with chloroform according to the Bligh & Dyer method. The solvent is distilled off from the extract under reduced pressure and the residue is purified to give long-chain fatty acids of high purity, useful as a starting substance for oleochemicals in industry, efficiently.

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 a long chain fatty acid by a microorganism belonging to the genus Francisella. Among the long-chain fatty acids produced by the method of the present invention, particularly arachidic acid having 20 or more carbon atoms, setreic acid, behenic acid, erucic acid, lignoceric acid, nervonic acid, ximenoic acid,
It is important as a raw material for oleochemicals.

[0002]

2. Description of the Related Art Bacteria of the genus Vibrio (Arch Microbiol. 1992, Vol. 157, 223-228) and Mycobacterium genus have been known as bacteria producing long-chain fatty acids having 20 or more carbon atoms. Bacteria (J. Biol. Chem. 1973, 248, 2303-
2309), a bacterium of the genus Francisella (J. Clinical Microbiol.
1989, Volume 27, 1601-1608) and the like are known. But,
The content of long chain fatty acids in the lipids produced by these bacteria is extremely low. Further, as the fatty acids having 20 to 24 carbon atoms, those extracted from fish oil are industrially used, but it is difficult to obtain a desired long-chain fatty acid with high purity.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for easily and efficiently producing long chain fatty acid.

[0004]

Means for Solving the Problems As a result of intensive studies on the method for efficiently producing long-chain fatty acids using microorganisms, the present inventors have found that one strain belonging to the genus Francisella is
They have found that they have an extremely high ability to produce long-chain fatty acids, and completed the present invention. That is, the present invention is a method for producing a long chain fatty acid, which comprises culturing Francisella species WYSA-40-2 strain and collecting the long chain fatty acid from the culture.

The present invention will be described in detail below. Examples of the strain used in the present invention include Francisella sp. WYSA-40-2 strain. Francisella sp. WYSA-40-2 strain is a strain newly isolated from the natural world, and its bacteriological properties are as follows. The morphological observation is
Francisella sp. WYSA-40-2 strain was inoculated into the medium and incubated at 30 ° C.
After culturing for 24 to 48 hours, it was performed using an optical microscope and a transmission electron microscope. The medium is Marine agar (Bacto
Marine Agar 2216, manufactured by Difco) or marine broth (Bacto Marine Broth 2216, manufactured by Difco) was used. In addition, since the growth of this strain is not observed when the concentration of sodium chloride in the medium is 1% or less, the biochemical properties test was carried out either by using 75% artificial seawater or by adding 3% in the medium. This was done by adding% sodium chloride.

A. Morphology 1) Cell shape and size: cocci, 0.4-0.8 μm 2) Polymorphism of cells: none 3) Motility, flagellation status: none 4) Spores: None 5) Gram stainability: negative

B. Growth condition in each medium 1) Marine agar plate culture: Good growth, colonies are round, convex, full-edge, wet light, opaque milky white 2) Marine agar slope culture: Good growth, filamentous, milky white 3) Marine broth culture: Produces good growth, turbidity and precipitation 4) Marine broth gelatin stab culture: liquefy (30 ° C,
1 week later, 20 ° C, 3 weeks later) 5) Litmus milk: The upper part of the medium is slightly alkalized, neither coagulated nor digested.

C. Physiological properties 1) Nitrate reduction: No reduction 2) MR test: − 3) VP test: + 4) Indole production: − 5) Hydrogen sulfide production: + w (using lead sugar paper) 6) Starch Hydrolysis: -7) Use of citric acid: Simmons Medium Not used: Cr
istensen medium Not used 8) Use of inorganic nitrogen source: Used 9) Pigment formation: None 10) Urease activity: Negative 11) Oxidase activity: Negative 12) Catalase activity: Positive 13) Growth range (temperature, pH): It grows best at temperatures of 10-45 ℃ and 34 ℃. pH 3-11, optimum growth pH range 6-
8 14) Attitude toward oxygen: Aerobic 15) OF test: Oxidation

16) Presence or absence of generation of acid and gas from saccharides. Basal medium uses 50% marine broth at 30 ° C. for 3 weeks. −: Not generated +: Generated 17) Esculin degradation: Negative 18) Arginine degradation: Negative DNA degradation: Degradation Poly-β-hydroxybutyric acid accumulation: No accumulation 19) Halophilicity: 1-10% 20 ) β-galactosidase: Negative 21) GC content: 32.8% 22) Isoprenoid quinone: Ubiquinone Q-8 23) Fatty acid composition (30 ° C): mol% 10: 0 (17.8%), 12: 0 (2.5%), 14 : 0 (4.1%), 16: 0
(5.0%), 18: 0 (6.0%), 18: 1 (13.1%), 20: 0 (6.3%),
20: 1 (1.8%), 22: 0 (14.6%), 22: 1 (3.4%), 24: 0
(6.1%), 24: 1 (19.1%)

Based on the above-mentioned mycological properties, comparison was made with known bacterial species in accordance with the classification criteria of Vergiz Manual of Determinative Bacteriology 8th Edition. This strain is an aerobic gram-negative coccus, has no flagella, and is oxidase-negative, catalase-positive, and aerobically produces acid from glucose in the OF test. A genus of Gram-negative aerobic coccus was searched from these characteristics, and it was considered that this strain corresponds to the genus Francisella because the GC content of DNA is low.

Furthermore, since the sodium salt was required for growth, the production of hydrogen sulfide, and the bacterial cell fatty acid composition were almost the same as those of the Francisella spp., This strain was identified as Francisella spp.
cisella sp.). This strain was designated as Francisella sp. WYSA-40-2, and deposited on November 9, 1994, at the Institute of Biotechnology, Institute of Industrial Science, as FERM P-14621.

As the strain used in the present invention, not only the above-mentioned Francisella species WYSA-40-2 strain but also a method for artificially mutating this strain, for example, ultraviolet irradiation, X-ray irradiation,
Mutant strains produced by treatment with mutagens or naturally occurring mutants that produce long-chain fatty acids can be used in the present invention. Next, a method for culturing the strain of the present invention will be described.

In the culture of the present invention, an ordinary bacterial culture method can be used. As the medium, either synthetic medium or natural medium can be used as long as it contains an appropriate amount of an assimilable carbon source, nitrogen source, inorganic substance and necessary growth and production promoting substances. As the carbon source, glucose, starch, dextrin, mannose, fructose, sucrose, lactose, xylose, arabinose, mannitol, molasses, etc. may be used alone or in combination. Further, ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soybean powder, casamino acid, etc. may be used alone or in combination depending on the assimilation ability of the bacterium. In addition, add inorganic salts such as sodium chloride, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, zinc sulfate, and copper sulfate as needed. . Further, a trace amount component that promotes the growth of the used bacterium and the production of long-chain fatty acid can be appropriately added.

The liquid culture method is most suitable as the culture method. The culturing temperature is suitably 10 ° C. to 45 ° C., and the pH of the medium during culturing is preferably maintained at 3 to 11, particularly 6 to 8 by adding calcium carbonate or the like. Usually 1 in liquid culture
After culturing for about 7 days, the target substance long-chain fatty acid is produced and accumulated in the cells. The culture is stopped when the maximum production in the culture is reached. Isolation and purification of long-chain fatty acids from the culture is carried out according to a method commonly used for isolating and purifying microbial metabolic products from the culture. For example, the culture is divided into a culture filtrate and cells by filtration, and the cells are extracted with chloroform, methanol or the like. Examples of the present invention will be shown below.

[0015]

[Example] Francisella sp. WYSA-40-2 strain was inoculated into 100 ml of a marine broth medium (manufactured by Difco) having the composition shown in Table 1, shake culture was carried out at 20 ° C for 2 days, and the cells were recovered by centrifugation. And lyophilized.

[0016]

[Table 1]

The dried bacterial cells are pulverized and then Bligh & Dyer method (Can J. Biochem. Physio
l. 37, 11 (1959) ) was extracted and purified by, the solvent was distilled off under reduced pressure to obtain a crude lipid 1.3 mg. Culture temperature 30 ℃ and 37
It was possible to obtain 1.2 mg and 1.3 mg of lipids by changing the temperature to ℃, culturing in exactly the same manner, and extracting. After partially methylating the lipid, the fatty acid composition was analyzed by gas chromatography (analysis conditions are as follows). The results are shown in Table 2. In addition, for the purpose of comparison Francisella Torarenshisu (Francise
The fatty acid composition of known strains belonging to Lla tularensis ) is shown in Table 3.
(J. Clin. Microbiol. 27 , (1989) 1601-1608). The peak obtained by gas chromatography is
Identification was performed by mass spectrometry.

Gas chromatography analysis conditions Column: Capillary column DB 23 (J & W
Internal diameter 0.25 mm Length 30 cm Column initial temperature: 150 ℃ Column final temperature: 210 ℃ Injection temperature: 270 ℃ Detector temperature: 300 ℃ Detector: FID Carrier gas: Helium

[0019]

[Table 2]

[0020]

[Table 3]

[0021]

INDUSTRIAL APPLICABILITY The present invention provides an efficient method for producing a long-chain fatty acid having high industrial utility.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI technical display location C12R 1:01) (72) Inventor Hiroshi Sano 1900 Sodeshicho, Shimizu City, Shizuoka Prefecture Kaiyo Bio Inc. Technology Research Center Shimizu Research Center

Claims (1)

[Claims] Claim 1. Francisella Species WYSA-40-2
A method for producing a long chain fatty acid, which comprises culturing a strain and collecting the long chain fatty acid from the culture.