JPH0698007B2 - Process for producing cyclopropane fatty acid by Pseudomonas bacteria - 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 cyclopropane fatty acid, which is extremely rare in nature, by culturing Pseudomonas bacteria in a medium containing propionic acid and the like.

[0002]

2. Description of the Related Art A fatty acid having a cyclopropane ring has been found in a lactic acid bacterium ( Lactobacillus arabinosus ) (Hofma
nn and Lucas, J. Am. Chem. Soc., 72, 4328-
4329, 1950), was named lactobacillic acid by lactic acid bacteria. This is shown as compound (1) below. After that, it has 19 carbon atoms, and the position of the cyclopropane ring is (n-7) (seventh carbon counting from the methyl group carbon) 2-hexyl-1-cyclopropane-1-decanoic acid (here, 19: Represented as 0 cp (n-7).)
Was confirmed. Furthermore, this fatty acid is contained in some bacteria such as Escherichia coli in addition to lactic acid bacteria, and in addition to lactobacillic acid, the following compound (2), 2-hexyl-1-cyclopropane-1-octanoic acid (17: 0 cp
(N-7)) was also found.

[0003]

[Chemical 1]

It was also revealed that these two cyclopropanoic acids were produced by using vaccenic acid (18: 1 (n-7)) and palmitoleic acid (16: 1 (n-7)) as precursors, respectively. (Law, Acc. Chem. Rec., 4
Vol., 199-203, 1971). That is, the reaction of these monoenoic acids with S-adenosylmethionine biosynthesizes cyclopropanoic acid having one more carbon atom. Characteristically, this reaction is specific to the (n-7) carbon. Cyclopropanoic acid has not been found in eukaryotic microorganisms such as yeast and cyanobacteria,
It has been reported that cyclopropane fatty acids with 19 carbon atoms were found in certain flagellates (Meyer and Holz, J. Biol. Che.
m., 241, 5000-5007, 1966.
Year). On the other hand, a fatty acid having a cyclopropene ring containing one double bond was found in seed oil of a plant of the family Asteraceae ( Sterculia faetide ) (Nunn, J. Chem. Soc., Pp. 313-3 ).
18, p. 1950), named after its name, sterculic acid (2-octyl-1-cyclopropene-1-octanoic acid) (here represented by 19: 1 cp (n-9)) (compound (3) below). Was named. Also mallow
( Malva sylvestris ), malvalic acid (2-octyl-1-cyclopropene-1-heptanoic acid), which is a C18 cyclopropene fatty acid, (18: 1 cp (n-
8)) (compound (4) below) was also found. The cyclopropane fatty acid corresponds to dihydrosterculic acid obtained by reducing the cyclopropene fatty acid contained in the above plant seed oil. Cyclopropane fatty acid can be produced by culturing various bacteria such as Escherichia coli, Vibrio, Salmonella, Pseudomonas, etc., but all the acids produced are only the above-mentioned compounds (1) and (2).

[0005]

[Chemical 2]

Regarding the physiological activities of these fatty acids, ingestion of oils and fats containing them inhibits desaturase,
Increased stearic acid and decreased oleic acid have been reported (Reiser and Raju, Biochem. Biophys. Res.
Commun 17: 8-8, 1964). It was also reported that when lactobacillic acid was added to the medium, lactic acid bacteria did not produce unsaturated acids with 18 carbon atoms (Hofmann et al., J. Biol. C.
hem., 213, 349-355, 1957).
Cyclopropane fatty acid has been found to have a biotin-like growth-promoting effect in culture of lactic acid bacteria (Hofmann and Pano).
s, J. Biol. Chem., 210, 687-693, 1
954). In the culture of various bacteria, the precursor monoenoic acid decreased and cyclopropanoic acid increased from the late culture phase, that is, the late logarithmic growth phase, and the latter was found to reduce the fluidity of cell membranes compared to the former (Jarrel ,
Biechemistry, Vol. 22, pp. 5611-5619, 19
1983), which is consistent with the fact that a decrease in monoenoic acid and an increase in cyclopropanoic acid are observed as the temperature is lowered. In Escherichia coli and Pseudomonas bacteria, the influence of culture conditions other than temperature on the composition of cyclopropane fatty acids (the compounds (1) and (2)) has been clarified.
It has been reported that decreasing dissolved oxygen, decreasing pH and increasing salinity contribute to the increase in these cyclopropane fatty acid compositions.

[0007]

An object of the present invention is to provide a method for efficiently producing a specific cyclopropane fatty acid using Pseudomonas bacteria.

[0008]

Means for Solving the Problems The present inventors have conducted various studies to achieve the above object, and as a result, in cyclopropane fatty acid-producing bacteria, cyclopropane fatty acids other than the compounds (1) and (2) were selected. In the search for bacteria and culture conditions that may produce, one Pseudomonas bacterium
It was found that (Pseudomonas sp. ATCC 12085) assimilates alcohols such as ethanol. In addition, bacterial cells were disrupted by sonication, and the alcohol dehydrogenase activity of various alcohols was measured using the crude extract. As a result, propanol showed the highest activity. Therefore, the present inventors have conducted extensive studies to produce cyclopropane fatty acids other than the compounds (1) and (2) using the Pseudomonas bacterium, and as a result, added propanol and propionic acid to the medium. It was found that cyclopropane fatty acid, which is rare in the natural world, can be produced, and the present invention has been completed.

(1) That is, according to the present invention, a Pseudomonas bacterium is cultured in a medium containing propanol and / or propionic acid or a salt thereof, and 2-pentyl-1-cyclopropane-1-decanoic acid (18: 0 cp (n-6 ))
Or 2-heptyl-1-cyclopropane-1-octanoic acid (18: 0 cp (n-8)) is obtained, (2) The medium contains a salt of propionic acid A method for producing a cyclopropane fatty acid according to item (1), and (3) a method for producing a cyclopropane fatty acid according to item (2), wherein the salt of propionic acid is sodium propionate.
In the present invention, the composition of the medium is not particularly limited except that it contains propanol or propionic acid, but a normal medium for marine bacteria (peptone and yeast extract) can be used. The pH is usually 5-9, preferably 6-8. The concentration of propanol or propionic acid in the medium is preferably 0.5 to 1.0%. The amount of Pseudomonas sp. To be inoculated is not particularly limited, but is usually 1% by volume of the culture solution. The culture temperature is preferably 22 to 28 ° C.
The fatty acid obtained by the method of the present invention can be separated as follows. That is, cells are collected from the culture broth after the growth by the centrifugal precipitation method, and lipid extraction is performed with a chloroform-methanol (1: 2) solution. Hydrolyzes the extracted lipids,
Methyl ester is used to lower the boiling point. The target compound is purified by subjecting the obtained methyl ester mixture to distillation under reduced pressure. If the unsaturated acid and the saturated acid are separated by an operation such as a low temperature crystallization method or a urea addition method before being subjected to distillation under reduced pressure, the purification will be more effective. The obtained fatty acid methyl ester can be converted into the target fatty acid by hydrolyzing it and then acidifying it.

Next, a preferred embodiment of the present invention will be described. The above-mentioned Pseudomonas bacterium was added to a basic medium (1 g of artificial seawater, 3 g of peptone, 1 g of yeast extract, HE
When cultured in PES 200 mM pH 7.5) and an ethanol-containing medium (basic medium to which 10 g of ethanol was added), the final cell concentration of the latter was 5 times that of the former. Therefore, the alcohol dehydrogenase activity of this bacterium was measured. The cultured cells were disrupted by ultrasonic waves, and the cell debris was removed by centrifugation to prepare a protein solution having a constant concentration from the supernatant, which was used as a sample. A solution of this protein solution and NAD ⁺ was used as a reaction solution, the reaction was initiated by adding alcohol, and the produced NADH was measured by a colorimetric method. As a result, the maximum activity is shown by propanol, and when the value is 100%, ethanol is 88%,
Butanol was 57% and methanol was 0%. Next, the bacterium was cultured in a basic medium supplemented with 10 g / l of propanol. As a result, the dehydrogenase activity of propanol was high, but the final cell concentration remained twice as high as that of the basal medium and did not reach that of ethanol. On the other hand, as a result of examining the fatty acid composition in the bacterial cell lipid, it was found that 16: 0, 1
6: 1 (n-7), 17:00 cp (n-7), 18: 1
Five fatty acids of (n-7) and 19: 0 cp (n-7) were observed, whereas in the medium containing propanol, in addition to the above acids, 15: 0, 17: 0, 17: 1.
(N-8), 18: 0 cp (n-8), 18: 0 cp
A fatty acid of (n-6) was also recognized. A gas chromatogram showing the fatty acid composition in the microbial cell lipid of the Pseudomonas bacterium is shown in FIG. FIG. 1 (a) is a basal medium, and FIG.
(B) is a basal medium supplemented with 0.5% propanol. According to the following formula, fatty acid synthesis using propanol as a starting material synthesizes an odd-chain fatty acid having 15 and 17 carbon atoms to produce two monoenoic acids (17: 1 (n-6)
(Compound (5)) and 17: 1 (n-8)) (compound (6)) each have two 18-carbon cyclopropane fatty acids (18: 6 cp (n-6) (compound (7)) and 18 : 0cp (n-8)) (compound (8)) was produced.

[0011]

[Chemical 3]

These fatty acids were determined to be cyclopropane fatty acids from the fact that they were not hydrogenated and from the mass spectra of their picolinyl esters (FIGS. 2 and 3). That is, as shown in FIGS. 2 and 3, the strong peaks of the molecular weights of 372 and 275 and 247 indicate that they are cyclopropanoic acids of (n-6) and (n-8) (Harvey, Biomed. Mas.
s Spectrom., Vol. 11, pp. 187-192, 1984
Year). FIG. 2 shows fatty acid 18: 0 cp (n-6), and FIG. 3 shows fatty acid 18: 0 cp (n-8). One of these (18: 0 cp (n-8)) is a reduction product of malvalic acid found in plants (dihydromalvalic acid).

It was further found that the use of propionate instead of propanol produced the same cyclopropane fatty acids. As shown in the examples below, 1% of total fatty acids in a medium supplemented with 0.5% propanol.
8.8% 18: 0 cp (n-8) and 5.7% 18:
A maximum value of 0 cp (n-6) is obtained. That is, adding more propanol to the basal medium not only increases the required culturing time but also reduces the composition of the target substance. In the case of sodium propionate, the required culturing time was longer than that of propanol, and in the medium supplemented with 500 mM sodium propionate (about 5%), 14.5% of total fatty acid was 18: 0 cp (n-8). A maximum value of 18: 0 cp (n-6) of 6.7% was obtained. That is, propanol is 1/1 more than sodium propionate
Although the target compound was similarly produced at a low concentration of 0, on the other hand, when sodium propionate was used, 18: 0 cp (n
The production rate of -6) is higher than that of propanol. When sodium propionate is used in the present invention, the production ratio of 2-pentyl-1-cyclopropane-1-decanoic acid to 2-hebutyl-1-cyclopropane-1-octanoic acid is increased.

[0014]

EXAMPLES Next, the present invention will be described in more detail based on examples. Example 1 0.5% propanol (5 g / liter) was added to a basal medium (3 g of peptone, 1 g of yeast extract, 200 mM of HEPES, pH 7.5 in 1 liter of artificial seawater), and Pseu was added.
Domonas sp . (ATCC 12085) was inoculated and shake culture (25 ° C) in a flask was performed for 77 hours. As a result, 1
8: 0 cp (n-8) and 18: 0 cp (n-6) were produced, each having a ratio to total fatty acids of 18.
8% and 5.7%. Example 2 Pseudomonas was prepared by adding 1.0% propanol to the basic medium.
As a result of culturing sp. for 77 hours in the same manner as in Example 1, 18:
0 cp (n-8) and 18: 0 cp (n-6) were produced, each having a ratio to total fatty acids of 9.0.
% And 5.7%.

Example 3 Pseudomonas was prepared by adding 0.2% propanol to the basal medium.
As a result of culturing sp. for 54 hours in the same manner as in Example 1, 18:
0 cp (n-8) and 18: 0 cp (n-6) were produced, each having a ratio of 6.6 to total fatty acids.
% And 3.6%. Example 4 Pseudomonas was prepared by adding 2.0% propanol to the basic medium.
As a result of culturing sp. for 175 hours in the same manner as in Example 1, 1
8: 0 cp (n-8) and 18: 0 cp (n-6) were produced with a ratio of 4.6 to total fatty acids respectively.
% And 2.5%.

Example 5 500 mM sodium propionate was added to the basal medium,
Pseudomonas sp. Was cultured for 232 hours in the same manner as in Example 1, and as a result, 18: 0 cp (n-8) and 18: 0 cp (n-).
6) was produced and their ratios to total fatty acids were 14.5% and 6.7%, respectively. Example 6 300 mM sodium propionate was added to the basal medium,
Pseudomonas sp. Was cultured for 232 hours in the same manner as in Example 1, and as a result, 18: 0 cp (n-8) and 18: 0 cp (n-).
6) was produced and their ratios to total fatty acids were 11.3% and 6.6%, respectively. Example 7 100 mM sodium propionate was added to the basal medium,
Pseudomonas sp. Was cultured for 232 hours in the same manner as in Example 1, and as a result, 18: 0 cp (n-8) and 18: 0 cp (n-).
6) was produced and their ratios to total fatty acids were 8.1% and 6.7%, respectively.

Example 8 To the basal medium was added 10 mM sodium propionate, and Ps
As a result of culturing eudomonas sp. for 119 hours in the same manner as in Example 1, 18: 0 cp (n-8) and 18: 0 cp (n-) were obtained.
6) was produced and their ratios to total fatty acids were 7.7% and 4.7%, respectively. Example 9 2 mM sodium propionate was added to the basal medium, and Pseu was added.
As a result of culturing domonas sp. for 47 hours in the same manner as in Example 1, 18: 0 cp (n-8) and 18: 0 cp (n-6) were obtained.
Are produced, and 2.2 for each of those total fatty acids
% And 2.1%.

[0018]

INDUSTRIAL APPLICABILITY According to the present invention, in the culture of Pseudomonas bacteria, cyclopropane fatty acid, which is rare in nature, can be efficiently produced by adding propionate or propanol to the medium. 2-Pentyl-1-cyclopropane-1-decanoic acid and 2-heptyl-1-cyclopropane-1-octanoic acid obtained by the method of the present invention are useful as unsaturated enzyme inhibitors and bacterial growth promoters.

[Brief description of drawings]

FIG. 1 is a gas chromatogram showing the fatty acid composition in cell lipids of Pseudomonas bacteria.

FIG. 2 is a mass spectrum of picolinyl ester of fatty acid (18: 0 cp (n-6)) produced by Pseudomonas bacteria.

FIG. 3 is a mass spectrum of picolinyl esters of fatty acids (18: 0 cp (n-8)) produced by Pseudomonas bacteria.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Masakazu Yamaoka, 1st Higashi 1-1 Tsukuba City, Ibaraki Prefecture Institute of Industrial Science and Technology, Institute of Chemical Research (72) Tohro Nakahara 1st 1-1 Higashi Tsukuba City, Ibaraki Industrial Technology In the laboratory

Claims (3)

[Claims] 1. A Pseudomonas bacterium is cultivated in a medium containing propanol and / or propionic acid or a salt thereof to prepare 2-pentyl-1-cyclopropane-1-decanoic acid or 2-heptyl-1-cyclopropane-1-octane. A method for producing a cyclopropane fatty acid, which comprises obtaining an acid. 2. The method for producing cyclopropane fatty acid according to claim 1, wherein the medium contains a salt of propionic acid. 3. The method for producing a cyclopropane fatty acid according to claim 1, wherein the salt of propionic acid is sodium propionate.