Note: Descriptions are shown in the official language in which they were submitted.
~1~5~60
1 BACKGRO~ND OF THE INVENTION
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1. Field of the Invention
-
The present invention relates to a process for the
production of Coenzyme Q. More particularly, it is concerned with
a process for the production of Coenzyme Q which comprises
cultivating a microorganism belonging to genus Pseudomonas in a
culture medium to which at least one member selected from the
group consisting of isopentenyl alcohol, dimethyl allyl alcohol,
geraniol, isopentenyl acetate, dimethyl allyl acetate, geranyl
acetate and ~ -methyl crotonic acid is added to form Coenzyme Q
and obtaining it.
The temm "Coenzyme Q" used in the present invention is
generally meant 2,3-dimethoxy-5-methyl-1,4-benzoquinones con-
taining an isoprene side chain in the 6-position of the quinone
nucleus represented by the general formula,
O
H3CO~ CH3
H3COJ~CH2-CH= lC - CH ~tnH
0 CH3
The present invention is intended to provide Coenzyme Q repre-
sented by the above formula in which n=8,9 and 10, respectively;
that is Coenzyme Q8' Coenzyme Qg and Coenzyme Q1o.
Coenzyme Q is widely distributed in animals, plants and
microorganisms etc., and it plays an important role as a con-
stitutive element of the terminal electron transfer system.
2. Explanation of the Prior Art
Recently it has been clarified that Coenzyme Q exhibits
excellent medical and physiological activities to various
diseases. In particular, Coenzyme 21o is considered most valuable
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as a medicine since Coenzyme Q of human being is Coenzyme Qlo.
It can be considered that the way of obtaining Coenzyme
Q is to extract from the animal and plant tissue, or microorgan-
isms, and to synthesize chemically. It is, however, difficult
to produce Coenzyme Q by extracting from the animal or plant
tissue on a large scale. Also, it is difficult to produce
Coenzyme Q by organic synthesis because of a disadvantage in
yields. Thus these procedures are not satisfactory for industrial
ones. On the other hand, the procedure to extract from micro-
organisms has the possibility of being employed economically
according to the yields of the cells and Coenzyme Q.
It is known that microorganisms belonging to genus
Pseudomonas produce Coenzyme Q8' Qg and Qlo
Thus we searched the compounds that were able to marked-
ly increase the content of Coenzyme Q per unit cell when they were
added to the culture medium in comparison with the case that they
were not added. P-hydroxy benzoic acid, and acetic acid and its
salts are known to be able to increase the content of Coenzyme Q
per unit cell when added to the culture medium (Japanese Patent
Publication No. 20396/72). It is known that the isoprene side
chain of Coenzyme Q lS produced through geranyl- and farnesyl
~;~ pyrophosphate by the biosynthesis in which the condensation of
isopentenyl- and dimethyl allyl pyrophosphate is repeated. However,
since these precursors are difficult to permeate through the cell
membrane, no attempt to make increase the content o Coenzyme Q
by adding such precursors to the culture medium has been reported~
SUMMARY OF THE INVENTION
The present invention provides a process for producing
Coenzyme Q which comprises cultivating microorganisms belonging
to genus Pseudomonas in a culture medium to which at least one
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5~
1 member selec~ed from the group consisting of isopentenyl alcohol,
dimethyl allyl alcohol, geraniol, isopentenyl acetate, dimethyl
allyl acetate, geranyl acetate and ~ -methyl crotonic acid is
added, to produce Coenzyme Q and obtaining it.
In the present invention, the term "Coenzyme Q" means
Coenzyme Q8' Qg and Qlo, which are thought to be important from
the industrial standpoint.
DETAILED DESCRIPTION OF THE INVENTION
We found that isopentenyl alcohol, dimethyl allyl alcohol,
geraniol, isopentenyl acetate, dimethyl allyl acetate, geranyl
acetate, and ~-methyl crotonic acid were easily utiliæed by the
microorganisms belonging to genus Pseudomonas when added to the
culture medium and that they were able to markedly increase the
content of Coenzyme Q per unit cell.
Any of microorganisms belonging to genus Pseudomonas
and capable of producing Coenzyme Q, can be employed in the
present invention. For example, these microorganisms capable of
producing Coenzyme Qlo include Pseudomonas diminuta ATCC 11568
(IAM-1513), etc., those capable of producing Coenz~me ~9 include
20 Pseudomonas schuylkilliensis ATCC 31419 (IAM-1126), Pseudomonas
denitrificans ATCC 13867 (I~-12023), Pseudomonas olevorans
ATCC 8062 (IAM-150~), Pseudomonas putrefaciens ATCC 8071 (IAM-
1509) etc., and those capable of producing Coenzyme Q8 include
Pseudomonas rubescens ATCC 12099 (IAM-1510), Pseudomonas fulva
ATCC 31418 (IAM-1529), Pseudomonas putida ATCC 4359 (IAM-1506)
etc.
In the culture medium used in practice of the present
invention, sugars such as glucose, molasses, etc., and any other
carbon sources with which these microorganisms are able to utilize,
can be used as carbon sources. Inorganic nitrogen compounds such
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v ~
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., , ;
s~
1 as ammonium su~fate, ammonium chloride and the like, organic
nitrogen compounds such as corn steep liquor, e~tracts of fish
meat, peptone, yeast extract, and the like, etc., can be usea as
nitrogen sources. In addition, as inorganic salts, potassium
salts, sodium salts, magnesium salts, salts of phosphoric acid
and sulfuric acid and the like, are employed.
The cultivation is usually carried out by agitation
with air under the condition of pH 4 to 8, temperature 25 to
35C and period 10 to 50 hours.
The addition of isopentenyl alcohol, dimethyl allyl
alcohol) geraniol, isopentenyl acetate, dimethyl allyl acetate,
geranyl acetate and ~ -methyl crotonic acid according to the
present invention can be carried out by a desired procedure and
desired time. For example, all amounts of the additive are added
at the start Or at a desired growth stage during the cultivation,
or it is added little by little according to the state of ferment-
-~ ation. Further, the additive can be used singly or in combination
with the other additives. The amount of the additive being added
is usually 1 x 10 5 ~o 5 x 10 3 mole/liter (as a final concentra-
tion~, preferably 5 x 10 5 to 5 x 10 4 mole/liter.
After the cultivation, Coenzyme Q formed is extracted
from the cells and separated from other materials. For example,
the wet cells obtained by centrifugation are extracted with
hydrophilic solvent such as acetone and the like; Coenzyme Q-
containing fraction is then transferred into a solvent such as
petroleum ether and the like; and the Coenzyme Q-containing
fraction is subjected to fractional purification by use of alumina
column, etc., whereby Coenzyme Q can be isolated.
In the present invention, the identlfication of Coenzyme
Q is conducted by comparing the product of this invention with
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1 a standard sample by means of W spectrum, measurement of melting
point and reversed phase thin layer chromatography in which a
mixture of acetone:water (9~:5) is used as a solvent, and others.
The following examples are given to explain the present
invention in more detail, but the present invention is not
limited by them.
Example 1
In a 30 liter-jar fermentor was placed 15 liters of a
culture medium (pH 7.0) containing 0.05~ of KH2P04, 0.15% of
Na2HP04, 0.05% of MgSO4 7H20, 1% of glucose, 1% of peptone and
0.2~ of yeast extract. After sterilized with steam, 645 milli-
grams of isopentenyl alcohol dissolved in 10 milliliters of
ethanol was added. Pseudomonas schuylkilliensis ATCC 31419
(IAM-1126), which had been previously cultured in 500 milliliters
of the culture medium havin~ the same composition as described
above for 2~ hours, was inoculated in the above culture medium
and cultured ~or 24 hours with aeration of 15 liter/minute at 27C.
After the cultivation, the culture broth was centrifuged
whereby 418 grams of wet cells (87 grams as dry cells) were
obtained.
To the wet cells was added 2 liters of acetone, and the
extraction was made with stirring. Then the cells were separated
by centrifugation. This procedure was repeated two times more.
The acetone extracts were combined together and condensed under
reduced pressure to distill away the acetone. Thereafter the
remaining solution was extracted three times with each 1 liter
of petroleum ether respectively and the resulting petroleum ether
layers were combined. The combined petroleum ether layer was
washed with water, dried over and condensed under reduced pressure.
; 3~ The residual oil was dissolved in a small amount of petroleum
,~
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1 ether and subjected to alumina ~olumn chromatography by eluting
with a mixture of petroleum ether and ethyl ether. The solvent
was dis~illed away from the above obtained eluate containing
Coenzyme Q. The residual oil was dissolved in a small amount of
ethanol and allowed to stand in a refrigerator whereby crystals
of Coenzyme Qg were appeared. These crystals were recrystalyzed
from ethanol three times and 142.6 milligrams of crystals of
Coenzyme Qg were obtained.
On the other hand, the same cultivation as above was
conducted by use of 15 liters of a culture medium to which no
isopentenyl alcohol was added. In the same manner as above, 359
grams of wet cells (69 grams as dry cells) were obtained. Further
the same procedure as above was applied whereby 81.42 milligrams
of crystals of Coenzyme Qg were obtained.
Based upon the above data, the effect of isopentenyl
alcohol on the production of Coenzyme Qg was calculated. The
addition of isopentenyl alcohol to the culture broth increased
the yield of Coenzyme Qg per liter of the broth by 75%, and by
39% per gram of dry cells. Thus it was clearly confirmed that
the addition of isopentenyl alcohol was effective to increase
the yield of Coenzyme Qg.
Example 2
The procedure o~ Example 1 was followed except that 1.16
grams of geraniol was used in place of isopentenyl alcohol, and
390 milligrams of wet cells (68 grams as dry cells) were obtained.
The wet cells were subjected to the same procedure as described
in Example 1, and 104.7 milligrams of crystals of Coenzyme Qg
were obtained.
On the other hand, the same microorganism was cultivated
by use of the culture medium to which no geraniol was added, and
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1 327 grams of wet cells (63 grams as dry cells) were obtained.
From th~ cells 74.3 milligrams of crystals of Coenzyme Q9 were
obtained.
Based upon thè above data, the effèct of geraniol on
the production of Coenzyme Qg was compared. The addition of
geraniol to the culture medium increased the yield of Coenzyme Qg
per liter of the culture broth by 41% and by 30% per gram of the
dry cells.
Example 3
Pseudomonas rubescens ATCC 12099 (IAM-1510) was cultivated
in the same culture medium and in the same manner as described in
Example ~, and 410 grams of wet cells (73 grams as dry cells) were
obtained. These were treated in the same manner as describe~ in
Example 1 and 58.4 milligrams of Coenzyme Q8 were obtained.
On the other hand, the same microorganism was cultivated
in the same culture medium as above except that no geraniol was
added, and 390 grams of wet cells (74 grams as dry cells) were
obtained. From the cells, 44.4 milligrams of Coenzyme Q8 were
obtained.
Based upon the above data, the same comparison as in
Example 1 was made. The addition of geraniol to a culture
medium increasecl the yield of Coenzyme Q8 per liter of the culture
broth by 32~ and 33% per gram of dry cells.
Example 4
Pseudomonas diminuta ATCC 11568 (IAM~1513) was culti-
....
vated by the same procedure as in Example 1 except that sodiumacetate was used in place of glucose in the composition of the
medium and three 300 milligrams fractions of isopentenyl alcohol
were added separately (total amount 900 milligrams). Thus 380
grams o wet cells ~69 grams as dry cells) were obtained, which
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were then subjected to the same treatment as in Example 1, where-
by 30.5 milllgrams of crystals of Coenzyme Qlo were obtained.
On the other hand, the same microorganism was cultivated
in the culture medium to which no isopentenyl alcohol was added,
and 320 grams of wet cells (61 grams as dry cells) were obtained,
from which 19.8 milligrams of crystals of Coenzyme Qlo were
obtained.
Based upon the above data, the same comparison as
Example 1 was made. The addition of isopen~enyl alcohol increased
the yield of Coenzyme Qlo per liter of the culture broth by 54%
and 38% per gram of dry cells.
Example 5
The procedure of Example 1 was followed except that
Pseudomonas denitrificans ATCC 13867 (IAM-12023) was used as
~ . . .
microorganism and that two 1 gram portions of geraniol were added
separately to the culture medium (total 2 grams) at different
times of cultivation. Thus 395 grams of wet cells (76 grams as
dry cells) were obtained, which was then subjected to the same
treatment as in Example 1, whereby 63.1 milligrams of crystals of
Coenzyme Qg were obtained.
On the other hand, the same microorganism was culti-
vated in the culture medium to which no geraniol was added, and
328 grams of wet cells (63 grams as dry cells) were obtained,
from which 37.2 milligrams of crystals of Coenzyme Q9 were
obtained.
Based upon the above data, the same comparison as in
Example 1 was made. The addition of geraniol increased the
yield of Coenzyme Qg per liter of the culture broth by 69% and
by 41% per gram of dry cells.
30~
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1 Exam21e 6
The procedure of Example 1 was followed except that
Pseudomonas fulva ATCC 31~18 (IAM-1529) was used as microorganism
:
and that three portions of isopentenyl alcohol (total 3 grams)
were separately added at different times of cultivation. Thus
390 grams of wet cells (73 grams as dry cells) were obtained,
which were then subjected to the same treatment as in Example 1,
whereby 86.4 milligrams of crystals of Coenzyme Q8 were obtained.
On the other hand, the same microorganism was cultivated
in the culture medium to which no isopentenyl alcohol was added
and 350 grams of wet cells (67 grams as dry cells) were obtained,
from which 60.S milligrams crystals of Coenzyme Q8 were obtained.
Based upon the above data, the same comparison as in
Example 1 was made. The addition of isopentenyl alcohol increased
the yield of Coenzyme Q8 per liter of the culture broth by 43%
and by 31% per gram of dry cells.
Example 7
The procedure of Example 1 was followed except that
Pseudomonas ru~escens ATCC 12099 (IAM-1510) was used as micro-
organism and that 645 milligrams of dimethyl allyl alcohol was
added. Thus 430 grams of wet cells (80 grams as dry cells) were
obtained, which were subjected to the same treatment as Example 1,
whereby 86 milligrams of crystals of Coenzyme Q8 were obtained.
On the other hand, the same microorganism was culti-
vated in the culture medium to which no dimethyl allyl alcohol
was added and 390 grams of wet cells (76 grams as dry cells) were
obtained, from which 68 milligrams of crystals of Coenzyme Q8
were obtained.
- Based upon the above data, the same comparison as
Example 1 was made. By the addition of dimethyl allyl alcohol
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1 the yield of Coenzyme Q8 per liter of the culture broth was
increased by 26~ and by 20~ per gram of dry cells.
Exam~le 8
The procedure of Example 1 was followed except that
Pseudomonas fulva ATCC 31418 (IAM-1529) was used as microorganism
and that 750 milligrams of ~ -methyl crotonic acid was added.
Thus 540 grams of wet cells (105 grams as dry cells) were
obtained, which were subjected to the same treatment as Example 1,
whereby 102 milligrams of crystals of Coenzyme Q8 were obtained.
On the other hand, the same microorganism was culti-
vated in the culture medium to which no ~ ~methyl crotonic acid
is added and 525 grams of wet sells (103 grams as dry cells)
were obtained, from which 79 milligrams of crystals of Coe~zyme
Q8 were obtained.
Based upon the above data, the same comparison as in
Example 1 was made. The addition of ~-methyl crotonic acid
increased the yield of Coenzyme Q8 per liter of the culture broth
by 28~ and by 26% per gram of dry cells.
Example 9
The procedure of Example 1 was followed except that
Pseudomonas olevorans ATCC 8062 (IAM-1508) was used as microorgan~
ism and that 960 milligrams of dimethyl allyl acetate was added.
Thus 490 grams of wet cells (94 grams as dry cells) were obtained,
which were subjected to the same treatment as Example 1, whereby
96 milligrams of crystals of Coenzyme Qg were obtained.
Gn the other hand, the same microorganism was cultivated
i in the culture medium to which no dimethyl allyl acetate was added
; and 470 grams of wet cells t90 grams as dry cells) were obtained,
from which 68 milligrams of crystals of Coenzyme Qg were obtained.
Based upon the above data, the same comparison as Example
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1 1 was made. The addition of dimethyl allyl acetate increased the
yield of Coenzyme Qg per liter of the culture broth by 42~ and
by 34% per gram of dry cells.
Example 10
The procedure of Example 1 was followed except that
Pseudomonas schuylkilliensis ATCC 31419 ~IAM-1126) was used as
_ . .
the microorganism and that 1.47 grams of geranyl acetate was
added. Thus, 530 grams of wet cells (93 grams as dry cells) were
obtained, which were subjected to the same treatment as Example
1, whereby 120 milligrams of crystals of Coenzyme Qg were obtained.
On the other hand, the same microorganism was cultivated
in the culture medium to which no geranyl acetate was added and
525 grams of wet cells (90 grams as dry cells) were ~btained,
from which 98 grams of crystals of Coenzyme Qg were obtained.
Based upon the above data, the same comparison as
Example 1 was made. The addition of geranyl acetate increased
the yield of Coenzyme Qg per liter of culture broth by 23% and
by 19~ per gram of dry cells.
Example 11
The procedure of Example 1 was followed except that `~
Pseudomonas denitrificans ATCC 13867 (IAM~12~23) was used as
microorganism and that the mixture of 960 milligrams of isopen-
tenyl acetate and l.47 grams of geranyl acetate was added. Thus
480 grams of wet cells (96 grams as dry cells) were obtained,
which were subjected to the same treatment as Example 1, whereby
115 milligrams of crystals of Coenzyme ~ wer~ obtained.
On the other hand, the same microorganism was cultivated
in the culture medium to which the mixture was not added and 490
gxams of wet cells (98 grams as dry cells) were obtained, from
which 76 milligrams of crystals of Coenzyme Qg were obtained.
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1 ~ased upon the above data, the same comparison as
Example 1 was made. The addition of isopentenyl acetate and
geranyl acetate increased the yield of Coenzyme Q~ per liter of
the culture broth by 51% and by 54% per gram of dry cells.
Example 12
Pseudomonas diminuta ATCC 11568 (IAM-1513) was culti-
.
vated by the same procedure as in Example 4 except that the
mixture of 960 milligrams of isopentenyl acetate and 960 milli-
grams of dimethyl allyl acetate was added. Thus 495 grams of wet
cells (98 grams as dry cells) were obtained, which were subjected
to the same treatment as Example 1, whereby 53 milligrams of
crystals of Coenzyme Qlo were obtained.
On the other hand, the same microorganism was cultivated
in the culture medium to which the mixture was not added. Thus
480 grams of wet cells (94 grams as dry cells) were obtained,
from which 39 milligrams of crystals of Coenzyme Qlo were obtained.
Based upon the above data, the same comparison as
Example 1 was made. The addition of isopentenyl acetate and
dimethyl allyl acetate increased the yield of Coenzyme Qlo per
liter of culture broth by 36~ and by 32% per gram of dry cells.
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