JPH04258295A - Production of phosphatidylcholine and phosphatidylethanolamine comprising linoleic acid as one and only constituent fatty acid - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to a method for producing phosphatidylcholine and phosphatidylethanolamine, which contain linoleic acid as the only constituent fatty acid, using a bacterial strain belonging to the genus Phialophora.

0002

BACKGROUND OF THE INVENTION Phospholipids, which are complex lipids, have components such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidic acid. However, it is difficult to separate these phospholipid species, and even molecular species due to differences in constituent fatty acids, from each other.
Therefore, it was rarely used alone.

However, since phospholipids are useful in constructing liposomes and play many physiological roles on biological membranes, they are expected to be used as pharmaceuticals.

[0004] In particular, phosphatidylcholine and phosphatidylethanolamine, which have linoleic acid as their only constituent fatty acid, are precursors to prostaglandins and the like that play important physiological roles in living organisms.

[0005]Currently, lipids are extracted by alcohol extraction from bacterial cells obtained by culturing Pelicularia bacteria in a medium containing a surfactant, and phosphatidylcholine containing linoleic acid as the only constituent fatty acid is extracted from the lipids obtained. A method for producing phosphatidylethanolamine and phosphatidylethanolamine has already been proposed (see JP-A-64-86888).

[0006] However, no method has been known so far for producing phosphatidylcholine and phosphatidylethanolamine in which linoleic acid is the only constituent fatty acid, using bacteria of the genus Phialophora, which is completely different from bacteria of the genus Pelicularia.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel method for producing phosphatidylcholine and phosphatidylethanolamine using linoleic acid as the only constituent fatty acid.

[0008]

The present inventors have developed a microorganism that produces phosphatidylcholine and phosphatidylethanolamine with linoleic acid as the only constituent fatty acid in a culture medium containing assimilable carbon, nitrogen, and inorganic salt sources. A search was conducted. As a result, it was discovered that microorganisms of the genus Phialophora produce phosphatidylcholine and phosphatidylethanolamine, which have linoleic acid as the only constituent fatty acid, leading to the present invention.

[0009] The present invention produces linol from bacterial cells obtained by aerobically cultivating a strain belonging to the genus Phialophora in a medium containing assimilable carbon, nitrogen, and inorganic salt sources. The present invention relates to a method for producing phosphatidylcholine and phosphatidylethanolamine whose only constituent fatty acid is linoleic acid, which is characterized by collecting phosphatidylcholine and phosphatidylethanolamine whose only constituent fatty acid is linoleic acid.

The microorganism of the present invention that produces phosphatidylcholine and phosphatidylethanolamine having linoleic acid as its only constituent fatty acid belongs to the genus Phialophora. As an example, Phialophora cyclaminis WF3010 (Phialophora cyclaminis), which belongs to the genus Phialophora, was isolated from soil by the present inventors.
laminis WF3010) strain can be most effectively utilized in the present invention. The mycological properties of this strain are as follows.

(a) Single sycamores (2.0-2.5μ x 7.0-15.0μ) are grown from well-formed and branched aerial hyphae (2.0-2.5μ) to the hyphae. It grows at right angles, and its tip is a “funnel”
It has a color like that. Conidia (2.0-2.5μ)
The conidia are transparent and spherical, and are formed as a head-shaped, viscous conidial mass at the tip of the sycamore. No sexual reproductive organs are observed. (b) All culturing in various media was carried out at 30°C, and the color tone of colonies was described in accordance with the expression method of "Color Standard" of the Japan Color Research Institute.

(1) Czapek agar medium Growth was moderate, and the colony diameter was 45 mm after 2 weeks of culture. There are few aerial hyphae, and the hyphae spread out in the medium in a feather-like manner, and the periphery is amorphous. Young colonies are pale olive gray, but as they mature, they become olive gray to dark olive gray.The color of the underside is dark olive gray, and no water-soluble pigment is produced. Conidia are fully grown and attached to the aerial hyphae of mature colonies, and the amount formed is moderate.

(2) Potato dextrose agar medium Growth is rather slow, reaching a diameter of 30 mm after 2 weeks of culture, but the number of aerial mycelia is moderately large, grows fluffy, and the periphery is amorphous. Young colonies are pale olive gray, but as they mature, they become olive gray to dark olive gray.The color of the underside is dark olive gray, and no water-soluble pigment is produced. Very few conidia are observed even in mature colonies.

(c) Growth temperature The growth temperature range is 8 to 37°C, and the optimum growth temperature is 25 to 33°C. No growth is observed at 5°C or 40°C.

Based on the above mycological properties, this fungus is classified as a fungus, and since no sexual reproductive organs are observed, it belongs to the Deuteromycetes. The W. Gumbs (1971) Cephalosporium-Altige Schimmelpilze (“
Cephalosporium-artige Sch
As a result of a search using M.
B. Schole-Schwartz's paper on the genus Phialophora: Personia, vol. 6, 59-94 (1970) (
When compared with the bacterial species described in Personia, 6, 59-94), the characteristics of this bacteria were similar to those of Phialophora cyclaminis (Phialophora cyclaminis).
The WF3010 bacterium was identified as Phialophora cyclaminis because it matched the characteristics described in is).

[0016] The present inventors have identified this bacterium as Phialophora.
Cyclaminis WF3010 (Phialopho
RA cyclominis WF3010) was submitted to the Agency of Industrial Science and Technology/National Institute of Microbial Technology.
It has been deposited under the number -11475.

[0017] For the production of phosphatidylcholine and phosphatidylethanolamine, which have linoleic acid as the only constituent fatty acid, the above-mentioned strains are not limited. Any capable material can be used in the present invention.

[0018] The method for culturing the above-mentioned microorganisms is in principle similar to the culture method for general microorganisms, but it is usually preferable to carry out the culture under aerobic conditions such as a shaking culture method using liquid culture or an aerated agitation culture method. .

The medium used for culture may be any medium containing a nutrient source that can be used by microorganisms belonging to the genus Phialophora, and any of various synthetic, semi-synthetic, and natural media can be used. As for the medium composition, carbon sources such as glucose, sucrose, fructose, glycerin, dextrin, starch, molasses, corn steep liquor, and organic acids may be used alone or in combination. As a nitrogen source, Pharmamedia,
Peptone, meat extract, yeast extract, soy flour, casein,
Organic nitrogen sources such as amino acids, urea, and inorganic nitrogen sources such as sodium nitrate and ammonium sulfate may be used alone or in combination.

Sodium salts, potassium salts, magnesium salts, phosphates, and other heavy metal salts may be added as required. In addition, when foaming is significant during culture, various known antifoaming agents can be added to the medium as appropriate, but it is necessary that the addition does not adversely affect the production of the target substance.

[0021] The pH of the medium is desirably within the normal optimum pH range for molds, for example 4.0 to 7.5. The culture temperature is a temperature at which microorganisms can grow well, usually 20 to 40°C.
It is particularly preferable to maintain the temperature around 30°C. In the case of liquid culture, the culture time is generally about 1 to 5 days. The desired substance is produced and accumulated through the above culture. Of course, the above-mentioned various culture conditions can be changed as appropriate depending on the type and characteristics of the microorganism used, external conditions, etc., and optimal conditions are selected and adjusted from the above ranges according to each.

[0022] To collect phosphatidylcholine and phosphatidylethanolamine, which have linoleic acid as the only constituent fatty acid, the above-mentioned phosphatidylcholine and the like are separated from the bacterial cells by a known separation method, such as an extraction method, and then isolated and purified by a known purification method. This is done by Preferably, the extraction method is carried out using alcohol. As the alcohol solvent, lower alcohols such as methanol, ethanol, and propanol are usually used, and methanol or ethanol is preferably used. In the extraction process, alcohol is added to the moist bacterial cells obtained from the culture solution by filtration or centrifugation in a weight ratio of 5 to 15, preferably 9 to 12, and the mixture is stirred at room temperature with a stirrer. This can be done effectively by stirring using a . It is appropriate that the extraction time be 2 to 4 hours. The alcoholic extract from the bacterial cells thus obtained is separated from the solid components of the bacterial cells by a filtration method or a centrifugation method.

After concentrating this alcoholic extract, the aqueous solution from which the alcohol has been removed is subjected to n-butanol extraction or the like to extract the target lipid into an organic solvent layer. Next, the extract is further subjected to chromatography using, for example, a silica gel column or a Sephadex LH-20 column (manufactured by Pharmacia) to isolate and purify phosphatidylcholine and phosphatidylethanolamine, respectively.

The two substances thus obtained are L-α-phosphatidylcholine dilinoleoyl (manufactured by Wako Pure Chemical Industries, Ltd.) and L-α-phosphatidylethanolamine dilinoleoyl (AVANTI POLAR-LIPIDS), respectively.
By comparing the retention time in HPLC analysis and the spectra of IR, NMR, MS, etc., with a standard product (manufactured by INC), it can be identified that these are phosphatidylcholine and phosphatidylethanolamine, each of which has linoleic acid as its only constituent fatty acid.

[0025]

[Effects of the Invention] By the method of the present invention, phosphatidylcholine and phosphatidylethanolamine, each of which has linoleic acid as its only constituent fatty acid, which is a precursor to brostaglandin, which plays an important physiological role in the body, are produced. can do.

[0026]

EXAMPLES Next, the present invention will be explained in more detail with reference to Examples. Note that percentages in the culture medium indicate weight/volume % unless otherwise specified.

Example 1 One platinum loop from the slant culture of strain WF-3010 (potato dextrose agar medium) was added to 100 ml of seed medium (potato starch 2%, glycose 1%, soybean flour 2%, phosphoric acid 1 potassium 0.1 %, magnesium sulfate 0.05%, pH not adjusted) into a 500 ml Erlenmeyer flask,
A seed culture solution was obtained by culturing with shaking at 28°C for 3 days. Add 200 ml of this seed culture to 100ml containing 5 L of medium with the same composition as above.
Two L-sized jar fermenters were inoculated. Culture was carried out at 28°C for 88 hours with aeration and stirring (aeration rate 5L/mi).
n, stirring at 300 rpm).

[0028] After the completion of culturing, approximately 10 L of the culture solution was filtered,
A solid content of bacterial cells was obtained. 10 L of methanol was added to the bacterial cells, and the mixture was stirred at room temperature using a stirrer for 4 hours to extract lipids. The methanol extract of the bacterial cells was concentrated under reduced pressure to remove methanol, and the resulting aqueous solution was about 400 ml.
was extracted twice with 400 ml of butanol to extract phosphatidylcholine and phosphatidylethanolamine into the butanol layer.

800 ml of the butanol extract was concentrated under reduced pressure to about 80 ml, and applied in two 40 ml portions to a Sephadex LH-20 (manufactured by Pharmacia) column (3 cm diameter, 45 cm) filled with methanol in advance. and eluted with 400 ml of methanol. Collect 8 ml of the eluate, develop each fraction with silica gel TLC (Merck Art 5715) (developing solution: chloroform/methanol = 5:2, sulfuric acid color development), and compare the Rf with the standard sample. , phosphatidylcholine and phosphatidylethanolamine were detected. As a result, fraction numbers 27-32
Confirm the elution of phosphatidylcholine and phosphatidylethanolamine and collect these fractions.
It was concentrated to dryness under reduced pressure.

Next, the dried product was dissolved in 5 ml of a chloroform-methanol mixture (1:1), and silica gel (Wako Gel C-200, Wako Pure Chemical Industries, Ltd.) filled with the same mixed solvent (chloroform/methanol = 1:1) was prepared in advance. company)
The column was attached to a column (3 cm diameter, 47 cm), and phosphatidylethanolamine was eluted with 900 ml of the same mixed solvent (chloroform/methanol = 1:1). After elution of phosphatidylethanolamine, methanol 900
Elution of phosphatidylcholine was performed in ml. The eluate was collected in 8 ml portions, and each fraction was developed on silica gel TLC in the same manner as before to detect phosphatidylcholine and phosphatidylethanolamine.

The eluted fractions of phosphatidylcholine and phosphatidylethanolamine were collected, concentrated to dryness under source pressure, and applied again to a Sephadex LH-20 column. In the case of phosphatidylethanolamine, the dried product is mixed with chloroform-methanol (1:2).
Dissolve in 6 ml of the same solvent (chloroform/
Sephadex LH- filled with methanol = 1:2)
20 column (3 cm diameter, 47 cm) and eluted with 400 ml of the same solvent (chloroform/methanol = 1:2). In the case of phosphatidylcholine, the dried product was dissolved in 6 ml of methanol and added to a Sephadex LH-20 column (3 cm diameter, 44 cm
) and eluted with 400 ml of methanol. Phosphatidylcholine and phosphatidylethanolamine were similarly detected in each eluted fraction by silica gel TLC analysis, and each eluted fraction was collected, concentrated under reduced pressure, and dried to remove phosphatidylcholine.
9mg, 157mg phosphatidylethanolamine
I got each.

Example 2 Regarding the phosphatidylcholine obtained in Example 1, L-α
- The physical and chemical properties were compared with a standard product of phosphatidylcholine dilinoleoyl (manufactured by Wako Pure Chemical Industries, Ltd.). As a result, IR of the obtained phosphatidylcholine, 1H-NM
The R, 13C-NMR and FAB-MS spectra completely matched those of the standard product, confirming the structure.

Furthermore, analysis conditions for HPLC analysis: Column: YMC-Pack ODS A-312 6
φ×150mm Mobile phase: Acetonitrile: Methanol (
1:1) Flow rate: 2.0 ml/min Detection: 210 nm It showed a retention time of 8.64 minutes, which was consistent with the standard.

Example 3 For the phosphatidylethanolamine obtained in Example 1, L-α-phosphatidylethanolamine dilinoleoyl (AVANTI POLAR-LIPIDS I
The physical and chemical properties were compared with a standard specimen (manufactured by NC). As a result, the IR of the obtained phosphatidylethanolamine
, 1H-NMR, 13C-NMR, and FAB-MS spectra completely matched those of the standard product, and the structure was confirmed.

Furthermore, analysis conditions for HPLC analysis: Column: YMC-Pack ODS A-312 6
φ×150mm Mobile phase: Acetonitrile: Methanol (
1:1) Flow rate: 2.0 ml/min Detection: 210 nm It showed 9.21 min, which was consistent with the standard.

Claims (2)

[Claims] Claim 1: A method of culturing a bacterial strain belonging to the genus Phialophora in a nutrient medium, and collecting phosphatidylcholine having linoleic acid as its only constituent fatty acid from the resulting bacterial cells, wherein linoleic acid is the only constituent fatty acid. A method for producing phosphatidylcholine. Claim 2: A method of culturing a bacterial strain belonging to the genus Phialophora in a nutrient medium, and collecting phosphatidylethanolamine having linoleic acid as its only constituent fatty acid from the obtained bacterial cells. A method for producing phosphatidylethanolamine as a fatty acid.