Note: Descriptions are shown in the official language in which they were submitted.
1340~33
PROCESS FOR PRODUCTION OF ARACHIDONIC ACID
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a new
process for the production of arachidonic acid.
2. Description of the Related Art
Known processes for the production of
arachidonic acid are those using microorganisms,
i.e., Penicillium, Aspergillus, Rhodotorula or
Fusarium, as disclosed in Japanese Examined Patent
Publication Nos. 56-19231, 56-19232, and 56-19233.
These processes, however, have the
disadvantages of a low yield, long term fermentation,
and a complicated production process.
However, a process for the production of
arachidonic acid using a microorganism belonging to
the genus Mortierella is now known.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a
new process for the production of arachidonic acid,
comprising culturing a microorganism belonging to the
genus Mortierella capable of producing arachidonic
acid to produce arachidonic acid or a lipid
comprising arachidonic acid, and recovering the
arachidonic acid.
DESCRIPTION OF THE PREFERRED EMBODIMENT
In the present invention, as a producer
microorganism, any strain belonging to the genus
Mortierella capable of producing arachidonic acid can
be used. For example, Mortierella elongate IFO 8570,
Mortierella exigua IFO 8571, and Mortierella
hygrophila IFO 5941 can be used. These strains are
stored in the Osaka Institute for Fermentation; 17-
85, Juso-honmachi 2-chome, Yodogawa-ku, Osaka 532,
Japan, and are available to the public without
limitation.
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Moreover, a new strain Mortierella elongata SAM
0219 can be used. This strain was newly isolated
from soil and identified by the present inventor, and
was deposited with the Fermentation Research
Institute, Agency of Industrial Science and
Technology (FRI), Higashi 1-1-3, Yatabe-cho, Tsukuba-
gun, Ibaraki-ken, Japan as FERM P-8703 on March 19,
1986, and transferred to International deposition
under the Budapest Treaty as FERM BP-1239 on December
22, 1986.
The above-mentioned new strain SAM 0219 (FERM
BP-1239) has the following taxonomical properties:
Cultural characteristics on various culture media
Culture condition: 25~C in the dark
1. Malt extract agar medium
Colonies growing fast, attaining a diameter
of 28 to 31 mm in two days and a diameter of 65 to
72 mm in five days; colonies are lobed; the formation
of aerial mycelium is scanty; sporulation is good,
sporangiophores arising from the aerial hyphae; the
mycelium has a garlic-like odor.
2. Potato dextrose agar medium
Colonies growing fact, attaining a diameter
of 27 to 31 mm in two days and a diameter of 75 to
mm in five days; colonies form a rosette pattern
of dense lobes; much aerial mycelium is formed at the
center of the colony; the reverse side of the colony
is yellowish white or yellow in color; sporulation is
poor; the mycelium has a rather strong garlic-like
odor.
3. Czapek's agar medium
Colonies growing moderately fast, attaining
a diameter of 22 to 24 mm in two days and a diameter
of 50 to 53 mm in five days; the formation of aerial
mycelium is scanty; occasionally, the aerial hyphae
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cling tightly to each other; sporulation is abundant;
the mycelium has a garlic-like odor.
4. LCA agar medium (prepared according to
Koichiro Miura and Mitsuyo Y. Kudo, "An agar-medium
for aquatic Hyphomycetes" Transactions of the
Mycological Society of Japan, vol. 11, p. 116-118,
1970).
Colonies growing fast, attaining a diameter
of 27 - 29 mm in two days and a diameter of 64 to 66
mm in five days; colonies are lobed; the formation of
aerial mycelium is scanty, except at the center of
the colony; sporulation is good; sporangiophores
arising from the aerial hyphae; the mycelium has a
garlic-like odor.
Microscopic Examination
Sporangiophore, mode of branching
sporangiophore, sporangium, sporangiospore, etc.,
were microscopically observed for microscopic
preparations and the colony per se from various
media.
A sporangiophore tapers and has a length of 87.5
to 320 um, a width of 3 to 7.5 ~m at the root, and a
width of 1.0 to 2.5 ~m at the top. A sporangiophore
often branches at the root. A sporangium is spherical
in form, has a diameter of 15 to 30 ~m, contains many
ascospores therein, and has an unclear color after
the detaching of the sporangiospore. A
sporangiospore is elliptical or, rarely, renal in
form, has a smooth surface, and a size of 7.5 to 12.5
x 5 to 7.5 ~m. A relatively large number of
chlamydospores are formed. Chlamydospores are
present separately or, rarely, linked in a chain
form. Occasionally, several mycelia appear from the
edge of the chlamydospore. Chlamydospore is
elliptical or subspherical in form, and has a size of
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12.5 to 30 x 7.5 to 15 ,um, or a diameter of 12.5 to
15 ,um. Zygospores are not observed.
Physiological Properties
Optical growth condition:
pH: 6 to 9,
Temperature: 20~C to 30~C;
Range for growth:
pH: 4 to 10;
Temperature: 5~C to 40~C.
On the basis of the above-mentioned taxonomical
properties, and according to J.A. von Arx, "The
Genera of Fungi Sporulating in Pure Culture" 3rd ed.,
J. Cramer, 1981; and K.H. Domsch, W. Gams and T.H.
Anderson, "Compendium of Soil Fungi", Academic Press,
1980, the strain SAM-0219 of the present invention is
considered to be a fungus belonging to the genus
Mortierella, because a sporangium is formed at a top
of a sporangiophore, sporangium has no collumella,
the sporangiospore has no appendage, and the mycelium
has a garlic-like odor.
Therefore, the taxonomical properties of the
strain of the present invention was compared with
those of known species of the genus Mortierella,
according to W. Gams, "A key to the species of
Mortierella, Persoonia 9: p381-391, 1977. As a
result, on the basis of the fact that the colony is
not velvety, the mycelium has a garlic-like odor, a
sporangiophore has a length of 87.5 to 320 um, and
branches at only its lower part and does not branch
racemousely, and a sporangium contains many
sporangiospores therein, the strain in question was
considered to fall under the genus Mortierella,
subgenus Mortierella, section Hygrophila. The
section Hygrophila includes 22 species. According to
a comparison of the present strain with these 22
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species, the present strain is similar to Mortierella
zychae, M. elongatula, and M. elongata.
Therefore, the strain of the present invention
was compared with the above-mentioned three strains,
referring to K.H. Domsch, W. Gams, and T.H. Anderson,
"Compendium of Soil Fungi", Academic Press, lg80; W.
Gams, "Some New or Noteworthy Species of
Mortierella"; Persoonia 9~ 140, 1976; G.
Linnemann, "Mortierella Coemans 1863"; H. Zyche and
R. Siepmann, "Mucorales Eine Beschreibung Aller
Gattungen und Arten dieser Pilzgruppe", p 155-241, J.
Cramer, 1965. The present strain is clearly
different from M. zychae in the length and width of
the sporangiophore at the base, and the size of the
sporangium. Moreover2, the present strain is
different from _. elongatula in the shape and size of
the sporangiospore. The present strain is different
from M. elongata in that sporangiophore is rather
shorter, the chlamydospore is ellipsoidal or
subglobose in form, rarely chlamydospores are linked
to each other in a chain form, and give rise to a
small number of radiating hyphae. However, the
present inventors concluded that such differences
between the present strain and M. elongata are not
sufficient to distinguish the present strain from _.
elongata, and thus identified the strain of the
present invention as Mortierella elongata, and
designated it as strain SAM 0219.
Spores, mycelia, or a preculture is used as an
inoculam for culturing the present strains. The
medium used may be a liquid or solid medium. A
liquid medium contains as a carbon source, for
example, glucose, fructose, xylose, saccharose,
maltose, soluble starch, molasses, glycerol, or
mannitol. Nitrogen sources include organic
substances such as peptones, yeast extract, meat
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extract, casamino acid, corn steep liquor, and
inorganic substances such as sodium nitrate, ammonium
nitrate, ammonium sulfate, and the like. If
necessary, inorganic salts such as phosphate salts,
magnesium sulfate, ferrous sulfate and cupric
sulfate, and vitamins may be included in a medium.
The concentration of these components is selected so
that such components do not adversely affect the
growth of the microorganism used. Practically, the
concentration of carbon source is 0.1 to 30% by
weight, preferably 1 to 10% by weight, relative to
the total weight of the medium. The concentration of
the nitrogen source is 0.01 to 5% by weight,
preferably 0.1 to 2% by weight, relative to the total
weight of the medium.
To enhance the production of arachidonic acid,
in addition to the above-mentioned medium components,
hydrocarbons, fatty acids or salts thereof, or fats
are preferably added to a medium in an amount of
0.01% to 20%. Hydrocarbons are preferably added to a
medium at the start of culturing, and fatty acids or
salts thereof are preferably added at the start of
and/or during culturing. When such an additive is
used during culturing, it is added at one time,
stepwise, or continuously.
The culturing temperature ranges 5~C to 40~C,
preferably 20~C to 30~C. A pH value of the medium is
4 to 10, preferably 6 to 9.
Culturing is preferably carried out with
aeration and/or agitation, with shaking in a liquid
medium, or with standing, and is usually carried out
for 2 to 10 days.
When culturing is carried out on a solid medium,
the solid medium is composed of wheat bran, chaff or
rice bran supplemented with water in an amount of 50
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to 100% by weight relative to the wheat bran, chaff
or rice bran.
If necessary, the medium is supplemented with a
small amount of nitrogen source, inorganic salts,
and/or minor nutrients.
Culturing is carried out at a temperature of 5~C
to 40~C, preferably 20~C to 30~C, for 3 to 14 days.
During culturing, lipid containing arachidonic
acid are intracellularly accumulated. When a liquid
medium is used, arachidonic acid is recovered from
the cultured cells by the following procedure.
After culturing, cultured cells are collected
from the cultured broth by a conventional means such
as filtration or centrifugation, the cells are washed
with water, and preferably, the washed cells are
dried. Drying is carried out by, for example,
lyophilization or air-drying. The dried cells are
treated with an organic solvent or a mixture thereof,
preferably under a nitrogen stream, to extract lipid
containing arachidonic acid. The organic solvent or
mixture thereof is, for example, ethers such as ethyl
ether, hydrocarbons such as hexane, alcohols such as
methanol or ethanol, halo-hydrocarbon such as
chloroform or dichloromethane, petroleum ether, as
well as a mixture of chloroform, methanol and water,
or a combination of methanol and petroleum ether
alternately used. By distilling off the solvent, a
lipid containing concentrated arachidonic acid is
obtained.
Alternatively, wet cells can be subjected to
extraction. In such a case, a water-miscible solvent
such as methanol or ethanol, or a water-miscible
solvent comprising the water-miscible solvent and
water or other organic solvent is used. The
extraction procedure is the same as described for
dried cells.
13~0~3~
The lipid thus obtained contains arachidonic
acid in the form of a lipid compound such as fat.
Although the arachidonic acid can be isolated in the
form of a free acid, it is preferably isolated in the
form of an ester with a lower alcohol, for example,
as methyl arachidonate. By converting arachidonic
acid to such an ester, it is easily separated from
other lipid components, and from other fatty acids
formed during culturing, such as palmitic acid, oleic
acid, linoleic acid and the like, which are also
esterified at the same time as the arachidonic acid
is esterified. To obtain methyl arachidonate, for
example, the lipid prepared as described above is
treated with a 5 to 10% hydrochloric acid solution in
absolute methanol or a 10 to 50% BF3 solution in
methanol for 1 to 24 hours at room temperature.
The mixture thus obtained is extracted with an
organic solvent such as hexane, ethyl ether or ethyl
acetate, to recover methyl arachidonate. Next, the
extract is dried over anhydrous sodium acetate, and
the solvent is distilled under reduced pressure to
obtain a residue mainly comprising a fatty acid
mixture. The mixture contains, in addition to the
target compound, methyl arachidonate, methyl
palmitate, methyl stearate, methyl oleate and the
like. From the mixture, methyl arachidonate is
isolated by column chromatography, low temperature
crystallization, a urea-adducting method, or a
combination thereof.
The isolated methyl arachidonate is then
hydrolyzed with an alkali and extracted with an
organic solvent such as ethyl ether, ethyl acetate,
or the like to obtain a free arachidonic acid.
Alternatively, arachidonic acid can be obtained,
without conversion to methyl ester, by alkalolysis
with, for example, 5% sodium hydroxide at a room
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temperature for 2 to 3 hours, followed by extraction
of the fatty acids from the alkalolysis product and
isolation of the target arachidonic acid.
Examples
The present invention will now be further
illustrated by, but is by no means limited to, the
following examples.
Example 1.
50 ml of a medium containing 5% glucose, 0.5%
peptone, 0.3% yeast extract and 0.3% malt extract (pH
6.0) was prepared and charged into a 500 ml-volume
Sakaguchi flask, and the whole was autoclaved for 20
minutes at 120~C. After cooling, Mortierella
elongata SAM 0219 (FERM BP-1239) was inoculated in
the medium, and then cultured for 5 days at 28~C with
reciprocal shaking at 110 rpm. After culturing, the
cultured broth was filtered to recover cells. The
cells were then completely washed with water and
lyophilized to obtain 1.3 g of dried cells. The
cells were extracted with a mixture of chloroform,
methanol and water, according to the one phase
extraction method, described by E.G. Bligh and W.J.
Dyer in Can. J. Biochem, Physiol., vol. 37, p. 911
(1959), to obtain 320 mg of the whole lipid. The
lipid was treated with a mixture of methanol and
hydrochloric acid (95:5) at 20~C for three hours to
esterify the arachidonic acid. The reaction mixture
was extracted with ethyl ether to obtain 200 mg of a
mixture of fatty acid methyl esters. The mixture
contained 9% methyl palmitate, 2% methyl stearate,
32% methyl oleate, 9% methyl linoleate, 10% methyl
~-linolenate , 21% methyl arachidonate and 17% other
components, as determined by gas chromatography. The
mixture was separated by column chromatography using
octa decylsilane with elution by 95% acetonitrile
solution to obtain fractions containing methyl
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arachidonate. After the fractions were combined, the
solvent was distilled off on a rotary evaporator to
obtain 25 mg of purified methyl arachidonate. The
methyl arachidonate preparation thus obtained was
compared with a commercially available authentic
methyl arachidonate preparation, by gas
chromatography, high performance liquid
chromatography, and mass spectrometry. Both
preparations showed the same results, revealing that
the preparation prepared in this Example is in fact
methyl arachidonate. The amount of methyl
arachidonate before and after the purification per
cultured broth was 0.84 mg/ml and 0.50 mg/ml
respectively; and those per dried cells were 32 mg/g
and 19 mg/g respectively.
Example 2
5 e of a medium having the same composition as
described in Example 1 was charged in a 15 e-volume
jar fermenter, and the medium was sterilized at 120~C
for 40 minutes. After cooling, the fermenter was
inoculated with 200 ml of a preculture of Mortierella
elongata SAM 0219 (FERM BP-1239). Culturing was
carried out at 30~C for 3 days with aeration of 0.5
v.v.m. The cultured broth was then filtered to
obtain 360 g of wet cells and 4350 e of a filtrate.
The cells were dried to obtain 110 g of dried cells.
The dried cells thus obtained were subjected to
extraction, hydrolysis and methyl-esterification
according to the same procedures as described in
Example 1, to obtain 29 g of whole lipid containing
18 g of a mixture of fatty acid methyl esters. The
mixture contained 8% methyl palmitate, 1% methyl
stearate, 29% methyl oleate, 12% methyl linoleate,
11% methyl ~-linolenate, 22% methyl arachidonate, and
17% other components, as determined by the same
procedure as described in Example 1. The amount of
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methyl arachidonate formed was 0.79 g/l broth, and 36
mg/g dried cells.
On the other hand, 4,350 ml of the above-
mentioned filtrate was subjected to extraction,
hydrolysis and methyl-esterification to obtain 156 mg
of a mixture of fatty acid methyl esters including
25% by weight of methyl arachidonate relative to the
weight of the mixture.
Example 3.
The same procedure as described in Example 1 was
carried out except that Mortierella exigua IFO 8571,
and Mortierella hygrophila IFO 5941 were used. 72 mg
and 95 mg of mixtures of fatty acid methyl esters
were obtained respectively, and from these mixtures,
12 mg and 20 mg of methyl arachidonate was isolated
and purified, respectively.
Example 4.
20 ml of a medium containing 2% glucose, 1~
yeast extract, and 0.2% Tween 20, as well as an
additive, i.e., 0.5~ of different kinds of
hydrocarbons, sodium salt of fatty acid or lipid
listed in the following Table 1 (pH 6.0 was charged
in each 100 ml-volume Erlenmeyer flask, and the
flasks were autoclaved at 120~C for 20 minutes.
Mortierella elongata SAM 0219 (FERM BP-1239) was
inoculated in the medium and then cultured for 5 days
at 28~C with rotary shaking at 200 rpm. The cultured
broths were separately filtered to obtain cells. The
cells were then subjected to extraction, hydrolysis
and methyl-esterification according to the same
procedure as described in Example 1. The weight of
the dried cells, amount of whole lipid, amount of
whole fatty acid methyl ester, content of methyl
arachidonate, and amount of methyl arachidonate per
cultured broth are set forth for each additive.
. ~
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13~0433
TABLE 1
Weight Amount of Amount of Content Amount of
Additive of driedwhole whole of methyl methyl
cellslipid fatty arachido- arachido-
(mg) (mg) acid natenate per
methyl (~) broth
esters (mg/mg)
(mg)
Octa-
decane 330 95 88 20 0.88
Sodium 290 81 64 25 0.80
Oleate
Sodium 300 96 83 19 0.79
linoleate
Olive oil 430 130 113 24 1.36
Corn oil 420 118 97 23 1.12
Coconut 380 98 78 25 0.98
oil
No 300 85 68 22 0.75
addition
As seen from the Table 1, the addition of
hydrocarbons, salts of fatty acids and lipid
increased the production of arachidonic acid by 10 to
80% relative to the no-addition control.
Example 5.
20 ml of a medium containing 2% glucose and 1%
yeast extract was charged in 100 ml-volume Erlenmeyer
flasks, and the flasks were autoclaved at 120~C for
20 minutes. Mortierella elongata SAM 0219 (FERM BP-
1239) was inoculated in the medium, and then
incubated at 28~C for 4 days. After the addition of
100 mg of a different kind of sodium salt of fatty
acid or lipid into each flask, incubation was
continued at 28~C for an additional 2 days. The
cultures were separately filtered to obtain cells.
The cells were then subjected to extraction,
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hydrolysis, and methyl-esterification according to
the same procedure as described in Example 1. The
amount of methyl arachidonate per dried cells and per
cultured broth was as set forth for each additive in
Table 2.
TABLE 2
AdditiveAmount of methyl arachi-
donate
mg/g dried mg/ml
cells broth
Sodium oleate 46 0.79
Sodium linoleate 47 0.80
Sodium linolenate 54 0.76
Olive oil 44 0.96
Soybean oil 53 1.12
Linseed oil 48 0.95
No addition 49 0.74
As seen from Table 2, the addition of salts of
fatty acids and lipids increased the production of
arachidonic acid by 10 to 60~ relative to the no-
addition control.
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