U.S. patent number RE28,886 [Application Number 05/366,263] was granted by the patent office on 1976-06-29 for method for preparing cytidine diphosphate choline.
This patent grant is currently assigned to Kyowa Hakko Kogyo Co., Ltd.. Invention is credited to Hiroshi Hagino, Kiyoshi Nakayama.
United States Patent |
RE28,886 |
Nakayama , et al. |
June 29, 1976 |
**Please see images for:
( Certificate of Correction ) ** |
Method for preparing cytidine diphosphate choline
Abstract
Cytidine diphosphate choline is prepared by culturing an aqueous
reaction liquor containing (a) at least one of the class consisting
of choline or phosphoryl choline; (b) at least one of the class
consisting of cytidine, cytidine-5'-monophosphate,
cytidine-5'-diphosphate or cytidine-5'triphosphate; (c) enzyme
systems of bacteria, yeasts or molds; (d) phosphate ion and (e) at
least one of the class consisting of magnesium ion and manganese
ion and recovering cytidine diphosphate choline from said culturing
liquor.
Inventors: |
Nakayama; Kiyoshi (Sagamihara,
JA), Hagino; Hiroshi (Tokyo, JA) |
Assignee: |
Kyowa Hakko Kogyo Co., Ltd.
(Tokyo, JA)
|
Family
ID: |
27297358 |
Appl.
No.: |
05/366,263 |
Filed: |
June 4, 1973 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
060743 |
Aug 3, 1970 |
03684652 |
Aug 15, 1972 |
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Foreign Application Priority Data
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Aug 4, 1969 [JA] |
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44-60999 |
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Current U.S.
Class: |
435/89; 435/839;
435/939; 435/931; 435/913; 435/886; 435/849; 435/872; 435/830;
435/840; 435/859; 435/876; 435/881; 435/911; 435/915; 435/935 |
Current CPC
Class: |
C07H
19/10 (20130101); C12P 19/305 (20130101) |
Current International
Class: |
C12P
19/00 (20060101); C07H 19/00 (20060101); C07H
19/10 (20060101); C12P 19/30 (20060101); C12D
013/06 () |
Field of
Search: |
;195/28N |
Other References
borkenhagen et al., The Journal of Biological Chemistry, vol. 227,
pp. 951-962, (1957). .
The Handbook of Enzymes, Asakura Shoten, pp. 374 & 375,
(1965)..
|
Primary Examiner: Tanenholtz; Alvin E.
Attorney, Agent or Firm: Fitzpatrick, Cella, Harper &
Scinto
Claims
What is claimed is: .[.1. A method for preparing cytidine
diphosphate choline, which comprises culturing an aqueous reaction
liquor containing (a) at least one of the class consisting of
choline of phosphoryl choline; (b) at least one of the class
consisting of cytidine, cytidine-5'-monophosphate,
cytidine-5'-diphosphate and cytidine-5'-triphosphate; (c) cells of
bacteria yeasts or molds that are in a form which liberate their
enzyme systems into the aqueous reaction liquor under the culturing
conditions; (d) phosphate ion (e) at least one of the class
consisting of magnesium ion and manganese ion (f) at least one
fermentable sugar and recovering cytidine diphosphate choline
from
said culturing liquor..]. 2. A method according to claim .[.1.].
.Iadd.14 .Iaddend.wherein said culturing is carried out at a
temperature of from
about 20.degree. C. to 55.degree. C. 3. A method according to claim
.[.1.]. .Iadd.14 .Iaddend.wherein the pH of said aqueous reaction
liquor
is from about 5.0 to 9.0. 4. A method according to claim .[.1.].
.Iadd.14 .Iaddend.wherein (a) comprises choline and (b)
comprises
cytidine-5'-monophosphate. 5. A method according to claim .[.1.].
.Iadd.14
.Iaddend.wherein (a) comprises choline and (b) comprises cytidine.
6. A method according to claim .[.1.]. .Iadd.14 .Iaddend.wherein
(a) comprises
choline and (b) comprises cytidine-5'-diphosphate. 7. A method
according to claim .[.1.]. .Iadd.14 .Iaddend.wherein (a) comprises
choline and (b)
comprises cytidine-5'-triphosphate. 8. A method according to claim
.[.1.]. .Iadd.14 .Iaddend.wherein said phosphate ion is supplied by
a mixture of
monopotassium phosphate and dipotassium phosphate. 9. A method
according
to claim .[.1.]. .Iadd.14 .Iaddend.(e) comprises magnesium sulfate.
10. A method according to claim .[.1.]. .Iadd.14 .Iaddend.wherein
said
fermentable sugar comprises glucose. 11. A method according to
claim .[.1.]. .Iadd.14 .Iaddend.wherein recovery of said cytidine
diphosphate choline is effected by passing a filtrate containing
the cytidine diphosphate choline obtained from the aqueous reaction
liquor through an
ion exchange resin column. 12. A method according to claim .[.1.].
.Iadd.14 .Iaddend.wherein (c) is at least one member of the class
consisting of:
Brevibacterium ammoniagenes ATCC 6872
Escherichia coli ATCC 21148
Serratia mercescens ATCC 2123
Aerobacter aerogenes ATCC 21217
Corynebacterium glutamicum ATCC 13287
Arthrobacter simplex ATCC 15799
Micrococcus sodonensis ATCC 11880
Pseudomonas fluorescens ATCC 21256
Bacillus subtilis ATCC 15512
Nocardia globerula ATCC 21022
Streptomyces ambofaciens ATCC 15154
Penicillium chrysogenum ATCC 15241
Aspergillus terreus ATCC 1012
Aspergillus flavus ATCC 13698
Gibberella fujikuroi ATCC 20136
Rhizopus delemar ATCC 20134
Mucor javanicus ATCC 15242
.[.saccharomyces cerevisiae ATCC 15248
Saccharomyces lactis ATCC 12425
Torulopsis sphaerica ATCC 8549
Zygosaccharomyces major (synonym Saccharomyces rouxii) ATCC
15249
Kloeckera africana ATCC 16512
Candida guilliermondii ATCC 9058.].
13. a method according to claim .[.1.]. .Iadd.14 .Iaddend.wherein
said fermentable sugar is present in a concentration from about 5
g./l. to 100 g./l. of said reaction liquor. .Iadd. 14. A method for
preparing cytidine diphosphate choline which comprises culturing an
aqueous reaction liquor containing (a) at least one of the class
consisting of choline and phosphoryl choline; (b) at least one of
the class consisting of cytidine, cytidine-5' -monophosphate,
cytidine- 5' -diphosphate and cytidine- 5' -triphosphate; (c) cells
of bacteria or molds that are in a form which liberate their enzyme
systems into the aqueous reaction liquor under the culturing
conditions; (d) phosphate ion (e) at least one of the class
consisting of magnesium ion and manganese ion; (f) at least one
fermentable sugar and recovering cytidine diphosphate choline from
said culture liquor.
Description
This invention relates to a method for preparing a high purity
cytidine diphosphate choline having utility as a biochemical
reagent, a medicinal preparation for treatment on head injury and
the like. An object of the present invention is to produce cytidine
diphosphate choline by a fermentation process efficiently and
economically in good yield, at low cost and on an industrial
scale.
As is known, cytidine diphosphate choline is a co-enzyme relating
to a lipid metabolism of living bodies. This compound can be
represented according to the following structural formula:
##EQU1##
Recently, it has been disclosed that cytidine diphosphate choline
can be separated from yeast (Science vol. 124, p. 81 (1956)) or
prepared according to a synthetic method (Journal of Biological
Chemistry vol. 222, pp. 185-191 (1956)). However, cytidine
diphosphate choline is difficult to produce on a commercial scale
since the yields obtainable with present methods of production are
unacceptably low leading to high production costs which may prove
prohibitive. Accordingly, despite its valuable utility, cytidine
diphosphate choline presently has little significance as a
practical matter. Thus, the importance of providing a method for
producing a purified preparate of cytidine diphosphate choline in
good yield at low cost and capable of implementation on an
industrial scale is clear.
In accordance with the present invention there is provided a method
adapted for use on an industrial scale for producing cytidine
diphosphate choline in good yield at low cost which comprises
culturing an aqueous reaction liquor i.e., nutrient medium,
containing (a) at least one of the class consisting of choline or
phosphorylcholine (b) at least one of the class consisting of
cytidine, cytidine-5' -monophosphate (hereinafter referred to as
"CMP"), cytidine--diphosphate (hereinafter referred to as "CDP")
and cytidine-5'-triphosphate (hereinafter referred to as "CTP");
(c) enzymes of at least one of the class consisting of yeast,
bacteria including Actinomycetes and molds; (d) phosphate ion and
(e) at least one of the class consisting of magnesium ion and
manganese ion. The culturing treatment can be carried out under
atmospheric pressure and normal temperature with conventional
equipment.
The starting raw materials, cytidine, CMP, CDP, or CTP are known
commercially available compounds and can be prepared according to
synthetic methods. In addition, cytidine or CMP may be prepared by
enzymatic decomposition of ribonucleic acid. CDP or CTP can also be
prepared by phosphation of CMP. Any of such materials can be used
herein either in the free form or nontoxic i.e., pharmaceutically
acceptable salt forms such as the sodium salt, barium salt,
etc.
Choline is produced on an industrial scale and is readily available
commercially. This material can be used in the form of its
pharmaceutically acceptable salts, e.g., chloride bromide,
citrates, gluconates, etc. Phosphorylcholine can be prepared by
phosphation of choline chloride, and is commercially available in
the form of the chloride and salt of chloride, like choline. Any of
the aforementioned forms can be used herein. In addition,
functional derivatives that serve as substitutes for choline and
phosphorylcholine can also be used.
The concentration of the choline, CMP, CDP, CTP, cytidine or
phosphorylcholine is not particularly critical apart from the
requirement that it be present in effective amounts, i.e., amounts
enabling the production of the quantity of product cytidine
diphosphate choline desired. In general, it is recommended to use
the cytidine CMP, CDP and/or CTP in amounts ranging from about 1
g./l. to 1,000 g./l., as a free acid concentration with a
concentration of 5 g./l. to 30 g./l. being preferred. The choline
and/or phosphorylcholine is preferably employed in a concentration
ranging from 0.5 g./l. to 50 g./l.
The phosphate ion requirements may be supplied by the addition of
inorganic phosphates such as KH.sub.2 PO.sub.4, K.sub.2 HPO.sub.4,
K.sub.3 PO.sub.4, Na.sub.2 HPO, NaH.sub.2 PO.sub.4, and Na.sub. 3
PO.sub.4 and the like, or alternatively, by the addition of the
free acid, e.g., phosphoric acid, which can then be neutralized
with suitable alkali such as NaOH, KOH, NH.sub.4 OH, or the like,
i.e., in situ formation of the phosphate. In certain instances,
e.g., when CTP and phosphorylcholine are used, it is not necessary
to add phosphate ion thereto as a separate additive, the requisite
phosphate ion being supplied by the CTP or phosphorylcholine.
The phosphate ion concentration is also not particularly critical
and may vary over a wide range depending upon the requirements of
the processor. In any event, for optimum results it is recommended
that the aqueous culturing medium contain from 2 g./l. to 200 g./l.
of phosphate ion as PO.sub.4 with a concentration of 10 g./l. to 40
g./l. being preferred.
The concentration of the magnesium ion and/or manganese ion
preferably supplied in the form of inorganic salts, e.g., magnesium
sulfate, manganese sulfate, etc., is preferably within the range of
0.001 g./l. to 3 g./l. In those instances where the enzyme is not
provided in a purified state, the magnesium or manganese are
supplied together with the enzyme preparate i.e., without any
special addition.
In any event, it will be understood that the aforedescribed ranges
are given only by way of defining preferred modes of proceeding and
thus, departures therefrom may be recommended in a particular
instance.
The yeast, bacteria and/or mold useful herein are provided in a
form such that enzyme systems of the yeast, bacteria and/or mold
cells can be readily liberated into the aqueous liquor under the
conditions employed for culturing. Suitable yeast, bacteria or mold
specimens may be prepared by drying, plasmolysis, treatment with a
solvent such as acetone or the like, extraction, surfactant
treatment, sonic or other treatment. Preferably, yeast, bacteria or
mold cell bodies obtained as by-products in industrial fermentation
and subjected to acetone-drying treatment, are recommended for use.
Extract liquor obtained by treating yeast, bacteria or molds
according to a mechanical rupture, sonic treatment, bacteriolysis
enzyme treatment, etc., can of course also be used. Various kinds
of bacteria, molds and yeasts can be used in the present invention
with specific representatives, including without necessary
limitation, the following:
Brevibacterium ammoniagenes ATCC 6872
Escherichia coli ATCC 21148
Serratia marcesens ATCC 2123
Aerobacter aerogenes ATCC 21217
Corynebacterium glutamicum ATCC 13287
Arthrobacter simplex ATCC 15799
Micrococcus sodonensis ATCC 11880
Pseudomonas fluorescens ATCC 21256
Bacillus subtilis ATCC 15512
Nocardia globerula ATCC 21022
Streptomyces ambofaciens ATCC 15154
Penicillium chrysogenum ATCC 15241
Aspergillus terreus ATCC 1012
Aspergillus flavus ATCC 13698
Gibberella fujikuroi ATCC 20136
Rhizopus delemar ATCC 20134
Mucor javanicus ATCC 15242
Saccharomyces cereviside ATCC 15248
Saccharomyces lactis ATCC 12425
Torulopsis sphaerica ATCC 8549
Zygosaccharomyces major (synonym, Saccharomyces rouxii) ATCC
15249
Kloecker africana ATCC 16512
Candida guilliermondii ATCC 9058
in accordance with a particularly preferred embodiment, there is
added to the aqueous reaction liquor at least one fermentable sugar
such as glucose, fructose, sucrose, maltose, etc. This enables the
production of even greater yields of cytidine diphosphate choline.
The fermentable sugar is preferably employed in a concentration
ranging from about 5 g./l.-100 g./l. of aqueous reaction
liquor.
After the reaction liquor is prepared by mixing the aforedescribed
components, the pH is adjusted to a value of 5.0-9.0, preferably
7.0-7.2 with suitable acid or alkali. Culturing may be carried out
at normal or slightly elevated temperatures, e.g., from
20.degree.-55.degree. C. until a substantial amount of cytidine
diphosphate choline is formed. The culturing time depends upon the
culturing temperature and pH, but generally requires about 0.5 to
10 hours. Formation of cytidine diphosphate choline can be traced
according to thin layer chromatography, or other suitable
analytical method.
After the completion of culturing, the bacteria, molds or solid
matters are removed from the culturing liquor, and then cytidine
diphosphate choline is separated and recovered according to the
well known procedure using an ion exchange resin as will explained
in detail in the examples which follow.
The followig examples are given for purposes of illustration only
and are not to be considered as necessarily constituting a
limitation on the invention.
Example 1
A 250 ml. conical flask containing 30 ml. of a reaction liquor (pH
7.0) having a composition of 7.38 mg./ml. of disodium salt of CMP,
24 mg./ml. of choline, 10 mg./ml. of glucose, 100 mg./ml. of
acetone-dried cells of Brevibacterium ammoniagenes ATCC 6872, 11.6
mg./ml. of monopotassium phosphate, 20 mg./ml. of dipotassium
phosphate and 2.96 mg/ml. of magnesium sulfate
was subjected to culturing at 30.degree. C. for 4 hours. Cytidine
diphosphate choline was formed and accumulated at a concentration
of 3.8 mg./ml. in the culture liquor.
The pH of 1.2 l. of filtrate containing 3.8 mg./ml. of cytidine
diphosphate choline, obtained by removing solid matters from the
culturing liquor, was adjusted to a pH of 8.5 with a 0.5 N KOH
solution. The filtrate was passed through a column of strongly
basic anion exchange resin, Dowex 1 .times. 2 (formic acid type).
After washing the resin with water, a formic acid solution was
passed through the column with gradual increase in the
concentration of formic acid (until 0.04 N max.). A fraction of
cytidine diphosphate choline was collected by elution according to
the so-called gradient elution method and absorbed onto carbon
powders. Then, elution was effected with acetone, and the eluate
was concentrated and dried. 1.3 g. of cytidine diphosphate choline
powders were obtained.
Example 2
Example 1 was repeated except that the glucose was omitted. 0.3
mg./ml. of cytidine diphosphate choline was obtained. When the
culturing was carried out in the reaction liquor further omitting
disodium salt of CMP which was substrate and choline, no cytidine
diphosphate choline was detected (less than 0.05 mg./ml.).
Example 3
Example 1 was repeated except that 4 mg./ml. of cytidine was used
in place of the disodium salt of CMP of Example 1. 0.5-0.6 mg./ml.
of cytidine diphosphate choline was obtained.
Examples 4-19
Example 1 was repeated except that acetone dried cell bodies of the
species shown in Table 1 were used in place of the cell bodies of
Brevibacterium ammoniagenes of Example 1. The results obtained are
as follows.
TABLE I ______________________________________ Ex. Amount.sup.1
number Strains (mg./ml.) ______________________________________ 4
Escherichia coli ATCC 21148 2.5 5 Serratia marcescens ATCC 21213
0.7 6 Aerobacter aerogenes ATCC 21217 0.6 7 Corynebacterium
glutamicum ATCC 13287 0.9 8 Arthrobacter simplex ATCC 15799 0.7 9
Micrococcus sodonensis ATCC 11880 0.5 10 Pseudomonas fluorescens
ATCC 21256 0.7 11 Bacillus subtilis ATCC 15512 0.6 12 Nocardia
globerula ATCC 21022 0.8 13 Streptomyces ambofaciens ATCC 15154 0.8
14 Penicillium chrysogenum ATCC 15241 0.9 15 Aspergillus terreus
ATCC 1012 0.6 16 Aspergillus flavus ATCC 13698 0.5 17 Gibberella
fujikuroi ATCC 20136 1.2 18 Rhizopus delemar ATCC 20134 0.5 19
Mucor javanicus ATCC 15242 0.5
______________________________________ .sup.1 Cytidine diphosphate
choline, formed.
Example 20
Example 1 was repeated except that 8.1 mg./ml. of CDP was used in
place of the disodium salt of CMP of Example 1. 4.1 mg./ml. of
cytidine diphosphate choline was obtained.
Example 21
Example 1 was repeated except that 9.7 mg./ml. of CTP was used in
place of the disodium salt of CMP of Example 1. 4.1 mg./ml. of
cytidine diphosphate choline was obtained.
Examples 22-37
Cytidine diphosphate choline was formed in amounts almost equal to
those of Examples 4-19 with each of the strains described therein
when culturing was carried out in 5 ml. of reaction liquor in a
test tube with shaking in the manner similar to that described in
Example 4-19, except that 8.1 mg./ml. of CDP or 9.7 mg./ml. of CTP
was used in place of CMP.
Example 38
A 250 ml. conical flask containing 30 ml. of a reaction liquor (pH
7.0) having a composition of 8.1 mg./ml. of CDP, 24 mg./ml. of
choline, 10 mg./ml. of glucose, 100 mg/ml. of dried baker's yeast,
11.6 mg./ml. of monopotassium phosphate, 20 mg./ml. of dipotassium
phosphate and 2.96 mg./ml. of MgSO.sub.4 .2H.sub.2 O was subjected
to culturing at 30.degree. C. for 4 hours. Cytidine diphosphate
choline was formed and accumulated at a concentration of 4.2
mg./ml. in the culture liquor.
The pH of 1.2 l. of the filtrate containing 4.2 mg./ml. of cytidine
diphosphate choline obtained by removing solid matters from the
culturing liquor, was adjusted to a pH of 8.5 with a 0.5 N KOH
solution. The filtrate was passed through a column of strongly
basic anion exchange resin, Dowex 1 .times. 2 (formic acid type).
After washing the resin with water, a formic acid solution was
passed through the column with gradual increase in the
concentration of formic acid (until 0.04 N max.). A fraction of
cytidine diphosphate choline was collected by elution according to
the so-called gradient elution method and absorbed onto carbon
powders. Then, elution was effected with acetone, and eluate was
concentrated and dried. 1.8 g. of cytidine diphosphate choline
powders were obtained.
Example 39
Example 38 was repeated except that the glucose was omitted. 0.4
mg./ml. of cytidine diphosphate was obtained. When the culturing
was carried out in the reaction liquor further free from CDP and
choline, no cytidine diphosphate choline was detected (less than
0.05 mg./ml.).
Example 40
Example 38 was repeated except that 9.7 mg./ml. of CTP was used in
place of the CDP of Example 38. 4.3 mg./ml. of cytidine diphosphate
choline was obtained.
Examples 41-46
Example 38 was repeated except that acetone dried cell bodies of
the species shown in Table 2 were used in place of the dried
baker's yeast of Example 38. The results obtained are as
follows:
TABLE II ______________________________________ Ex. Amount.sup.1
number Strains (mg./ml.) ______________________________________ 41
Saccharomyces cerevisiae ATCC 15248 3.7 42 Saccharomyces lactis
ATCC 12425 2.3 43 Torulopsis sphaerica ATCC 8549 2.8 44
Zygosaccharomyces major (synonym, Saccharomyces rouxii) ATCC 15249.
2.1 45 Kloeckera africana ATCC 16512 1.2 46 Candida guilliermondii
ATCC 9058 1.1 ______________________________________ .sup.1
Cytidine diphosphate choline, formed.
Examples 47-52
Examples 41-46 were repeated except the 9.7 mg./ml. of CTP were
used in place of CDP of Examples 41-46. The results are as
follows:
TABLE III ______________________________________ Ex. Amount.sup.1
number Strains (mg./ml.) ______________________________________ 47
Saccharomyces cerevisiae ATCC 15248 3.6 48 Saccharomyces lactis
ATCC 12425 3.3 49 Torulopsis sphaerica ATCC 8549 3.1 50
Zygosaccharomyces major ATCC 15249 1.7 (synonym, Saccharomyces
rouxii). 51 Kloeckera africana ATCC 16512 1.2 52 Candida
guilliermondii ATCC 9058 1.3 ______________________________________
.sup.1 Cytidine diphosphate choline, formed.
Example 53
A 250 ml. conical flask containing 30 ml. of a reaction liquor (pH
7.0) having a composition of 7.38 mg./ml. of disodium salt of CMP,
24 mg./ml. of choline, 10 mg./ml. of glucose, 100 mg./ml. of dried
baker's yeast, 11.6 mg./ml. of monopotassium phosphate, 20 mg./ml.
of dipotassium phosphate, 2.96 mg./ml. of MgSO.sub.4.2H.sub.2 O was
subjected to culturing at 30.degree. C. for 4 hours. Cytidine
diphosphate choline was formed and accumulated at a concentration
of 4.0 mg./ml. in the culture liquor.
The pH of 1.2 l. of filtrate containing 4.0 mg./ml. of cytidine
diphosphate choline obtained by removing solid matters from the
culture liquor, was adjusted to pH of 8.5 with a 0.5 N KOH
solution. The filtrate was passed through a column of strongly
basic anion exchange resin, Dowex 1 .times. 2 (formic acid type).
After washing the resin with water a formic acid solution was
passed through the column with gradual increase in the
concentration of formic acid (until 0.04 N max.). A fraction of
cytidine diphosphate choline was collected by elution according to
the so-called gradient elution method, and absorbed onto carbon
powders. Then, elution was effected with acetone, and the eluate
was concentrated and dried. 1.8 g. of cytidine diphosphate choline
powders was obtained.
Example 54
Example 53 was repeated except that the glucose was omitted. 0.4
mg./ml. of cytidine diphosphate choline was obtained. When
culturing was carried out in the reaction liquor further omitting
CMP and choline, no cytidine diphosphate choline was detected.
(less than 0.05 mg./ml.)
Examples 55-60
Example 53 was repeated except that acetone dried cell bodies of
the species shown in Table IV were used in place of the dried
baker's yeast of Example 53. The results obtained are as
follows:
TABLE IV ______________________________________ Ex. Amount.sup.1
number Strains (mg./ml.) ______________________________________ 55
Saccharomyces cereuisiae ATCC 15248 3.6 56 Saccharomyces lactis
ATCC 12425 2.2 57 Torulopsis sphaerica ATCC 8549 2.8 58
Zygosaccharomyces major ATCC 15249 2.1 (synonym, Saccharomyces
rouxii). 59 Kloeckera africana ATCC 16512 1.3 60 Candida
guilliermondii ATCC 9058 1.1 ______________________________________
.sup.1 Cytidine diphosphate choline, formed.
Example 61
A 250 ml. conical flask containing 30 ml. of a reaction liquor (pH
7.0) having a composition of 7.38 mg./ml. of disodium salt of CMP,
72.4 mg./ml. of phosphorylcholine, 10 mg./ml. of glucose, 100
mg./ml. of dried baker's yeast, 11.6 mg./ml. of monopotassium
phosphate, 20 mg./ml. of dipotassium phosphate, 2.96 mg./ml. of
MgSO.sub.4.2H.sub.2 O was subjected to culturing at 30.degree. C.
for 4 hours. Cytidine diphosphate choline was formed and
accumulated at a concentration of 3.8 mg./ml. in the culture
liquor.
The pH of 1.2 l. of filtrate containing 3.8 mg./ml. of cytidine
diphosphate choline obtained by removing solid matters from the
culture liquor, was adjusted to pH of 8.5 with a 0.5 N KOH
solution. The filtrate was passed through a column of strongly
basic anion exchange resin, Dowex 1 .times. 2 (formic acid type).
After washing the resin with water a formic acid solution was
passed through the column with gradual increase in the
concentration of formic acid (until 0.04 N max.). A fraction of
cytidine diphosphate choline was collected by elution according to
the so-called gradient elution method, and absorbed onto carbon
powders. Then, elution was effected with acetone, and the eluate
was concentrated and dried. 1.7 g. of cytidine diphosphate choline
powders was obtainded.
* * * * *