Title of Invention	METHOD OF PURIFYING CYTIDINE DIPHOSPHATE CHOLINE
Abstract	A method of purifying cytidine diphosphate choline, which comprises contacting a cytidine diphosphate choline solution containing a nucleic acid analogue and having a pH of not less than 0.5 and not more than 5.0 with an H-type strongly acidic cation exchange resin, and eluting cytidine diphosphate choline adsorbed onto the resin with water or an aqueous solution having an ion concentration of not more than 0.1 mol/L to separate and purify the cytidine diphosphate choline.

Title of Invention

METHOD OF PURIFYING CYTIDINE DIPHOSPHATE CHOLINE

Abstract

A method of purifying cytidine diphosphate choline, which comprises contacting a cytidine diphosphate choline solution containing a nucleic acid analogue and having a pH of not less than 0.5 and not more than 5.0 with an H-type strongly acidic cation exchange resin, and eluting cytidine diphosphate choline adsorbed onto the resin with water or an aqueous solution having an ion concentration of not more than 0.1 mol/L to separate and purify the cytidine diphosphate choline.

Full Text	DESCRIPTION METHOD OP PURIFYING CYTIDINE DIPHOSPHATE CHOLINE Technical Field [0001] The present invention relates to a method of purifying cytidine diphosphate choline useful as a starting material of pharmaceutical products and a starting material of nutritious foods. Background Art [0002] As the method of purifying cytidine diphosphate choline (hereinafter to be abbreviated as CDP-choline), chemical synthetic methods (patent reference 1, patent reference 2) and methods using a culture of a microorganism or an enzyme (patent reference 3, patent reference 4) are known. As the method of purifying CDP-choline produced by a chemical synthetic method, a method using an anion exchange resin (patent reference 1) and a method using both of a strongly acidic ion exchange resin and a weakly basic ion exchange resin (patent reference 2) are known. In the latter method, two kinds of ion exchange resins are used. Moreover, while phosphorylcholine and cytidine-5'-monophosphate (hereinafter to be abbreviated as CMP) contained in a CDP-choline solution can be separated, uracil or uridine-5'-triphosphate (hereinafter to be abbreviated as UTP) cannot be separated efficiently by this method. [patent reference l] Japanese Published Examined Patent Application No. 6558/1988 [patent reference 2] Japanese Published Examined Patent Application No.31306/1994 [patent reference 3] Japanese Patent No.3369236 [patent reference 4] WO99/49073 Disclosure of the Invention Problems to be Solved by the Invention [0003] 1 An object of the present invention is to provide a method of purifying CDP-choline, in which method CDP-choline can be separated from a nucleic acid analogue conveniently. Means of Solving the Problems [0004] The present invention relates to the following (1) - (6). (1) A method of purifying CDP-choline, which comprises contacting a CDP-choline solution containing a nucleic acid analogue and having a pH of not less than 0.5 and not more than 5.0 with an H-type strongly acidic cation exchange resin, and eluting CDP-choline adsorbed onto the resin with water or an aqueous solution having an ion concentration of not more than 0.1 mol/L to separate and purify CDP-choline. (2) The method of the above-mentioned (1), wherein the CDP- choline solution is prepared from a resulting medium containing CDP-choline, which has been produced and accumulated therein by placing a biocatalyst having an activity to produce CDP-choline from a precursor of UTP and choline or phosphorylcholine in an aqueous medium together with the precursor of UTP and choline or phosphorylcholine or a salt thereof. (3) The method of the above-mentioned (2), wherein the biocatalyst comprises a culture or a treated culture of a microorganism capable of producing UTP from a precursor of UTP, and a culture or a treated culture of a microorganism capable of producing CDP-choline from UTP and choline or phosphorylcholine. (4) The method of the above-mentioned (2), wherein the biocatalyst comprises an enzyme that catalyzes a reaction to produce CDP-choline from a precursor of UTP and choline or phosphorylcholine. (5) The method of the above-mentioned (4), wherein the enzyme that catalyzes a reaction to produce CDP-choline is an enzyme selected from the group consisting of orotate phosphoribosyl transferase, orotidine-5'-monophosphate decarboxylase, uridine 2 phosphorylase, uracil phosphoribosyltransferase, uridine kinase, uridylate and cytidylate kinases, nucleoside diphosphate kinase, cytidine-5'-triphosphate synthase, choline phosphate cytidyltransferase and choline kinase. (6) The method of any of the above-mentioned (1) to (5), wherein the nucleic acid analogue is selected from uracil and UTP. Effect of the Invention [0005] The present invention provides CDP-choline and a salt thereof at a low cost. Best Mode for Carrying Out the Invention [0006] The CDP-choline solution to be used in the present invention may be prepared by any method as long as it contains a nucleic acid analogue and has a pH of not less than 0.5 and not more than 5.0. When the prepared CDP-choline solution has a pH of 0.5 - 5.0, it is directly used. When the pH is higher than 5.0, an acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and the like, is added, and when the pH is lower than 0.5, an alkali such as sodium hydroxide, potassium hydroxide and the like is added to adjust to pH 0.5 - 5.0, preferably 1.0 - 3.5, before use. [0007] The H-type strongly acidic cation exchange resin may be of a gel type or a porous type, as long as it is an H-type strongly acidic cation exchange resin. Specific examples thereof include the Dowex series (e.g., HCR-S, HCR-W2, Marathon C, Monosphere 650C, MSC-1, Monosphere 88, 50W2, 50W4, 50W8 etc.) manufactured by Dow Chemical Company, Diaion SK series (e.g., SK1B, SK104, SK110, SK112 etc.) manufactured by Mitsubishi chemical, Diaion PK series (e.g., PK208, PK212, PK220, PK228 etc.) manufactured by Mitsubishi Chemical, Amberlite series (e.g., IR120B, IR124 etc.) manufactured by Rohm and Haas and the like. 3 [0008] Degree of crosslinking of the H-type strongly acidic cation exchange resin is not particularly limited as long as CDP-choline and nucleic acid analogue can be separated at the degree of crosslinking. It is preferably 2 - 10%, more preferably 4-6%. In the present invention, the H-type strongly acidic cation exchange resin is preferably used in the form of a packed column, and as the column to be used in the present invention, any column may be used. The CDP-choline solution containing a nucleic acid analogue and having a pH of not less than 0.5 and not more than 5.0 is contacted with the H-type strongly acidic cation exchange resin by applying the solution to a column packed with the resin and the like to allow adsorption of CDP-choline onto the resin. For example, when the solution is applied to a column packed with an H-type strongly acidic cation exchange resin having a degree of crosslinking of 2 - 10%, the flow rate (SV) is 0.1 - 5.0, preferably 0.2 - 3.0. [0009] By the above-mentioned adsorption treatment, a nucleic acid analogue contained in the solution, particularly a pyrimidine type nucleic acid substance such as orotic acid, orotidine-5'-monophosphate (hereinafter to be abbreviated as OMP), uridine-5'-monophosphate (hereinafter to be abbreviated as UMP), uridine-5'-diphosphate (hereinafter to be abbreviated as UDP), UTP, CMP, cytidine-5'-diphosphate (hereinafter to be abbreviated as CDP), cytidine-5'-triphosphate (hereinafter to be abbreviated as CTP) and the like can be efficiently separated or removed from CDP-choline. Out of the nucleic acid analogues, uracil and UTP are particularly preferably separated and removed, since they hardly adsorb to or do not at all adsorb to the resin, when a CDP-choline solution containing uracil and UTP is brought into contact with the resin at a pH of not less than 0.5 and not more than 5.0. 4 [0010] CDP-choline adsorbed on the H-type strongly acidic cation exchange resin is eluted by applying an aqueous solution having an ion concentration of not more than 0.1 mol/L, preferably not more than 0.03 mol/L, more preferably water, and the like, whereby CDP-choline is eluted from the resin, for the separation and purification. Where necessary, the CDP-choline-containing solution collected by the above-mentioned elution step may be subjected to an activated carbon treatment or decolorization treatment using a nonpolar porous synthetic adsorbent, for example, Diaion HP series (e.g., HP20, HP21 etc.) manufactured by Mitsubishi Chemical, Diaion SP800 series (e.g., SP825, SP850 etc.) manufactured by Mitsubishi Chemical, Diaion SP200 series (e.g., SP207 etc.) manufactured by Mitsubishi Chemical, Amberlite XAD series (e.g., XAD4, XAD7HP, XAD16HP, XAD1180, XAD2000 etc.) manufactured by Rohm and Haas and the like. [0011] The above-mentioned CDP-choline-containing solution or decolorized solution is adjusted to pH 2.0 - 4.0 with acid or alkali. After concentration is carried out as necessary, the concentration of CDP-choline is adjusted to 50 - 800 g/L, preferably 100 - 700 g/L, and crystals of CDP-choline can be obtained using an organic solvent, preferably a hydrophilic organic solvent such as acetone, ethanol, methanol, propanol and the like. In addition, the above-mentioned CDP-choline-containing solution or decolorized solution is adjusted to pH 5.0 - 9.5 with sodium hydroxide. After concentration is carried out as necessary, the concentration of CDP-choline is adjusted to 50 - 800 g/L, preferably 100 - 700 g/L, and crystals of CDP- choline sodium salt can be obtained using an organic solvent, preferably a hydrophilic organic solvent such as acetone, ethanol, methanol, propanol and the like. [0012] 5 Examples of the method of obtaining CDP-choline crystals using an organic solvent include a method comprising adding an organic solvent to a CDP-choline solution to allow precipitation of crystals, and a method comprising adding dropwise a CDP-choline solution to a large amount of an organic solvent to allow precipitation of crystals. In the present invention, the CDP-choline solution containing a nucleic acid analogue may be any as long as it is a solution containing CDP-choline and a nucleic acid analogue. Examples thereof include a solution produced by a chemical synthetic method, a method using a biocatalyst having an activity to produce CDP-choline from a precursor of UTP and choline or phosphorylcholine (hereinafter to be abbreviated as CDP-choline-producing activity). [0013] Examples of the biocatalyst include a culture of a microorganism having a CDP-choline-producing activity, a treated culture thereof, an enzyme that catalyzes a reaction to produce CDP-choline and the like. As the microorganism, any microorganism can be used as long as it has a CDP-choline-producing activity. A microorganism inherently having a CDP-choline-producing activity can be used as it is for the production of CDP- choline, and a microorganism naturally having no CDP-choline- producing activity can be used for the production of CDP- choline by introducing a DNA encoding an enzyme that catalyzes a reaction to produce CDP-choline from a precursor of UTP and choline or phosphocholine. Preferable examples of the microorganism include microorganisms belonging to the genera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, Streptococcus, Sinorhizobium, Haemophilus, Arthrobacter, Aureobacterium, Cellulomonas, Clavibacter, Curtobacterium, Pimerobacter, Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichia, Candida and the like. 6 [0014] Examples of the microorganism belonging to the genus Escherichia include microorganisms belonging to Escherichia coli such as Escherichia coli MM294, Escherichia coli XLl-Blue, Escherichia coli XL2~Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli GI698, Escherichia coli TBI and the like. Examples of the microorganism belonging to the genus Serratia include Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens and the like. Examples of the microorganism belonging to the genus Bacillus include Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens and the like. Examples of the microorganism belonging to the genus Brevibacterium include Brevibacterium immariophilum, Brevibacterium saccharolyticum, Brevibacterium flavum, Brevibacterium lactofermentum and the like. Examples of the microorganism belonging to the genus Corynebacterium include microorganisms belonging to Corynebacterium glutamicum such as Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC138 69 and the like, microorganisms belonging to Corynebacterium ammoniagenes such as Corynebacterium ammoniagenes ATCC6872, Corynebacterium ammoniagenes ATCC21170 and the like, microorganisms belonging to Corynebacterium acetoacidophilum such as Corynebacterium acetoacidophilum ATCC1387 0, etc. and the like. Examples of the microorganism belonging to the genus Microbacterium include microorganisms belonging to Microbacterium ammoniaphilum such as Microbacterium ammoniaphilum ATCC15354 and the like, Microbacterium lactium, Microbacterium imperiale and the like. Examples of the microorganism belonging to the genus Pseudomonas include Pseudomonas putida and the like. Examples of the microorganism belonging to the genus Streptococcus include Streptococcus pneumoniae and the like. Examples of the 7 microorganism belonging to the genus Sinorhizobium include Sinorhizobium meliloti and the like. Examples of the microorganism belonging to the genus Haemophilus include Haemophilus influenzae and the like. Examples of the microorganism belonging to the genus Arthrobacter include Arthrobacter citreus, Arthrobacter globiformis and the like. Examples of the microorganism belonging to the genus Aureobacterium include Aureobacterium flavescens, Aureobacterium saperdae, Aureobacterium testaceum and the like. Examples of the microorganism belonging to the genus Cellulomonas include Cellulomonas flavigena, Cellulomonas carta and the like. Examples of the microorganism belonging to the genus Clavibacter include Clavibacter michiganensis, Clavibacter rathayi and the like. Examples of the microorganism belonging to the genus Curtobacterium include Curtobacterium albidum, Curtobacterium citreum, Curtobacterium luteum and the like. Examples of the microorganism belonging to the genus Pimerobacter include Pimerobacter simplex and the like. [0015] Examples of the microorganism belonging to the genus Saccharomyces include Saccharomyces cerevisiae and the like. Examples of the microorganism belonging to the genus Schizosaccharomyces include Schizosaccharomyces pombe and the like. Examples of the microorganism belonging to the genus Kluyveromyces include Kluyveromyces lactis and the like. Examples of the microorganism belonging to the genus Trichosporon include Trichosporon pullulans and the like. Examples of the microorganism belonging to the genus Schwanniomyces include Schwanniomyces alluvius and the like. Examples of the microorganism belonging to the genus Pichia include Pichia pastoris and the like. Examples of the microorganism belonging to the genus Candida include Candida utilis and the like. [0016] 8 In addition, more preferable examples of the microorganism include microorganisms belonging to the genera Escherichia, Bacillus, Brevibacteriuirt, Corynebacterium and Saccharomyces, further preferably, microorganisms belonging to the genera Escherichia, Brevibacterium, Corynebacterium. [0017] Of the above-mentioned microorganisms, when a microorganism naturally having a CDP-choline-producing activity has only an insufficient CDP-choline-producing activity, a recombinant DNA having a DNA encoding an enzyme that catalyzes a reaction to produce CDP-choline from a precursor of UTP and choline or phosphorylcholine may be introduced into the microorganism according to a conventional method, or a cell of different microorganism having the activity may be fused therewith to prepare a microorganism having an enhanced activity. [0018] As the microorganism having an enhanced activity or a microorganism imparted with the activity, a transformant obtained by introducing a DNA encoding an enzyme that catalyzes the reaction into a microorganism according to the following method can be preferably used. Examples of the DNA encoding an enzyme that catalyzes a reaction to produce CDP-choline from a precursor of UTP and choline or phosphorylcholine (hereinafter to be abbreviated as a CDP-choline-producing enzyme) include DNAs encoding orotate phosphoribosyl transferase [EC 2.4.2.10] having an activity to produce OMP from orotic acid, orotidine-5'-monophosphate decarboxylase [EC 4.1.1.23] having an activity to produce UMP from OMP, uridine phosphorylase [EC 2.4.2.3] having an activity to produce uridine from uracil, uracil phosphoribosyltransferase [EC 2.4.2.9] having an activity to produce UMP from uracil, uridine kinase [EC 2.7.1.48] having an activity to produce UMP from uridine, uridylate and cytidylate kinases [EC 2.7.1.48] having an activity to produce 9 UDP from UMP, nucleoside diphosphate kinase [EC 2.7.4.6] having an activity to produce UTP from UDP, cytidine-5'- triphosphate synthetase [EC 6.3.4.2] (hereinafter to be abbreviated as PyrG) having an activity to produce CTP from UTP, choline kinase [EC 2.7.1.32] (hereinafter to be abbreviated as CKI) having an activity to produce phosphorylcholine from choline and choline phosphate cytidyltransferase [EC 2.7.7.15] (hereinafter to be abbreviated as CCT) having an activity to produce CDP-choline from CTP and phosphorylcholine and the like. [0019] Preferable examples of the DNA encoding a CDP-choline- producing enzyme include DNAs encoding PyrG, CKI and CCT. A DNA encoding PyrG has been cloned from the chromosome of Escherichia coli, and its entire nucleotide sequence has been determined [J. Biol. Chem., 261, 5568(1986)]. Examples of a recombinant having a DNA encoding PyrG include pMW6 [Biosci. Biotechnol. Biochem., _6_1, 956 (1997)], which is a plasmid having a 2426 bp NruI-PstI fragment containing a DNA encoding PyrG derived from Escherichia coli inserted into an Smal-PstI site of a multicloning site of vector pUC8 of Escherichia coli [Gene, 19, 259 (1982)] and the like. [0020] The entire nucleotide sequence of the DNA encoding CCT has been determined [Eur. J. Biochem., 169, 477(1987)]. Examples of a recombinant DNA having a DNA encoding CCT include plasmid pCC41 [Biochemical, 1296 bp Dral fragment containing a DNA encoding CCT derived from yeast inserted into a Smal site of a multicloning site of vector pUC18 of Escherichia coli [Gene, 33, 103(1985)] and the like. [0021] A DNA encoding CKI has also been cloned from a chromosome of yeast in the same manner, and the entire nucleotide sequence thereof has been determined [J. Biol. Chem., 264, 10 2053(1989)]. Examples of a recombinant DNA having a DNA encoding CKI include plasmid pCKID [J. Biol. Chem., 264, 2053(1989)] having a 2692 bp Pstl-Hindlll fragment containing a DNA encoding CKI derived from yeast inserted into a shuttle vector YEpM4 of yeast and Escherichia coli, [Mol. Cell. Biol., 1_, 29 (1987)] and the like. [0022] The above-mentioned plasmids can be isolated and purified from Escherichia coli having these plasmids, according to a known method [Nuc. Acids Res., 1_, 1513(1979)]. A biocatalyst having a CDP-choline-producing activity can be obtained by obtaining a DNA encoding a CDP-choline- producing enzyme from the plasmid obtained as mentioned above according to, for example, Molecular Cloning, A Laboratory Manual, Third Edition, Sambrook et al. edit, Cold Spring Harbor Laboratory (2001), incorporating the DNA into an expression vector to prepare a recombinant DNA, and transforming the above-mentioned microorganism as a host cell. [0023] First, a DNA encoding PyrG, CCT or CKI is obtained from the above-mentioned plasmid pMW6, plasmid pCC41 or plasmid pCKID and, based on the obtained DNA, a DNA fragment having a suitable length and containing a part encoding the polypeptide is prepared as necessary. Where necessary, moreover, a DNA wherein nucleotide of the nucleotide sequence of a partial DNA fragment corresponding to a CDP-choline-producing enzyme are substituted so as to provide a codon optimal for the expression of a host cell is prepared. The DNA is useful for an efficient production of a CDP-choline-producing enzyme. [0024] A recombinant vector is produced by inserting the DNA fragment or full-length DNA into the downstream of a promoter of a suitable expression vector. In this case, DNA encoding a CDP-choline-producing enzyme may be independently inserted 11 into an expression vector, or plural DNAs may be inserted into the same expression vector. The recombinant vector is introduced into a host cell suitable for the expression vector. [0025] As the host cell, the above-mentioned microorganisms can be mentioned. As the expression vector, one capable of autonomous replication or one capable of being incorporated into a chromosome in the host cell, and having a promoter at a site permitting transcription of a DNA encoding a CDP-choline- producing enzyme can be used. When prokaryote such as bacterium and the like is used as a host cell, a recombinant vector having a DNA encoding a CDP- choline-producing enzyme is preferably autonomously replicatable in a prokaryote, as well as constituted with a promoter, a ribosome binding sequence, the DNA and a transcription termination sequence. A gene regulating the promoter may also be contained. [0026] Examples of the expression vector include pBTrp2, pBTacl, pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by QIAGEN), pKYPlO (Japanese Published Unexamined Patent Application No. 110600/58), pKYP200 [Agric. Biol. Chem., 4£, 669 (1984)], pLSAl [Agric. Biol. Chem. , 5_3, 277 (1989)], pGELl [Proc. Natl. Acad. Sci. USA, 82_, 4306 (1985)], pBluescript II SK(-) (manufactured by Stratagene), pTrs30 [prepared from Escherichia coli JM109/pTrS30 (FERM BP- 5407)], pTrs32 [prepared from Escherichia coli JM109/pTrS32 (FERM BP-5408)], pGHA2 [prepared from Escherichia coli IGHA2 (FERM BP-400), Japanese Published Unexamined Patent Application No. 221091/60], pGKA2 [prepared from Escherichia coli IGKA2 (FERM BP-6798), Japanese Published Unexamined 12 Patent Application No. 221091/60], pTerm2 (US Patent 4686191, US Patent 4939094, US Patent 5160735), pSupex, pUBHO, pTP5, pCl94, pEG400 [J. Bacterid., 172, 2392 (1990)], pGEX (manufactured by Pharmacia), pET system (manufactured by Novagen) and the like. [0027] As the promoter, any promoter can be used as long as it is functionable in the host cell. Examples of the promoter include those derived from Escherichia coli, phage and the like, such as trp promoter (PtrP), lac promoter,' PL promoter, PR promoter, T7 promoter and the like. In addition, a promoter having an artificially altered design such as a promoter (Ptrp2) in which two Ptrp are sequently connected in tandem, tac promoter, lac T7 promoter and let I promoter, and the like can also be used. [0028] It is preferable to use a plasmid in which the distance between a Shine-Dalgarno sequence (ribosome binding sequence) and an initiation codon is adjusted to a suitable distance (e.g., 6 to 18 bases). In the recombinant vector of the present invention, a transcription termination sequence is not always necessarily for the expression of a DNA encoding a CDP-choline-producing enzyme. However, it is preferable to place a transcription termination sequence directly downstream the structure gene. [0029] As a method of introducing the recombinant vector, any method can be used as long as a DNA can be introduced into the above-mentioned host cell. For example, a method using a calcium ion [Proc. Natl. Acad. Sci. USA, 659, 2110 (1972)], a protoplast method (Japanese Published Unexamined Patent Application No. 248394/1988), the method described in Gene, 11_, 107 (1982) or Molecular & General Genetics, 1_68, 111 (1979) and the like can be used. [0030] 13 When a yeast is used as the host cell, for example, YEpl3 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15 and the like can be used as the expression vector. As the promoter, any promoter can be used as long as it permits expression in a yeast strain. Examples of the promoter include promoters of genes of the glycolytic system such as hexose kinase and the like, PH05 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MFal promoter, CUP 1 promoter and the like. [0031] As the method of introducing the recombinant vector, any method can be used as long as it introduces the DNA into a yeast. Examples thereof include an electroporation method [Methods Enzymol., 1_94, 182 (1990)], a spheroplast method [Proc. Natl. Acad. Sci. USA, 75_, 1929 (1978)], a lithium acetate method [J. Bacteriology, 153, 163 (1983)], the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and the like. [0032] When the microorganism has only a part of the CDP- choline-producing activity, two or more kinds of microorganisms may be combined as appropriate to afford a CDP- choline-producing activity, and used as the biocatalyst having a CDP-choline-producing activity. Even when the microorganism has a CDP-choline-producing activity, two or more kinds of microorganisms can be combined. Two or more kinds of microorganisms to be combined may be selected from any of the above-mentioned microorganisms. Examples of the combination include a combination of microorganisms belonging to the same genus or different genera selected from the microorganisms belonging to the genera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, Streptococcus, Sinorhizobium, Haemophilus, Arthrobacter, Aureobacterium, 14 Cellulomonas, Clavibacter, Curtobacterium, Pimerobacter, Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichia and Candida, and the like. [0033] For example, a combination of the microorganism belonging to the genus Corynebacterium and a microorganism belonging to the genus Escherichia and the like can be mentioned. Specific example is a combination of Corynebacterium ammoniagenes ATCC21170 and Escherichia coli MM294/pCKG55 strain (FERM BP- 3717) (Japanese Patent No. 3369236, US Patent No. 6387667) and the like. The example of the culture of a microorganism having a CDP-choline-producing activity, which is one of the biocatalysts having a CDP-choline-producing activity, is a culture obtained by culturing, according to a conventional method, the microorganism obtained by the above-mentioned method. [0034] When the microorganism is a prokaryote such as bacteria and the like or a eukaryote such as yeast and the like, any of a natural medium and a synthetic medium can be used as the medium used for culturing the microorganism as long as it contains a carbon source, a nitrogen source, inorganic salts and the like to be assimilated by the microorganism, and enables efficient culturing of the microorganism. Any carbon source may be used as long as the microorganism can assimilate it, and glucose, fructose, sucrose, molasses containing them, carbohydrates such as starch and starch hydrolysate, organic acid such as acetic acid, propionic acid and the like, alcohols such as ethanol, propanol and the like, and the like can be used. [0035] As the nitrogen source, ammonium salts of inorganic acid or organic acid such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate and the like, 15 other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean cake and soybean cake hydrolysate, various bacterial cells obtained by fermentation and digests thereof, and the like can be used. [0036] As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used. Culturing is performed under aerobic conditions by, for example, shaking culturing, aerobic stirred culturing and the like. The culturing temperature is preferably 15°C - 40°C and the culturing time is generally 16 hours - 7 days. The pH is preferably maintained 3-9 during culturing. The pH is adjusted with inorganic or organic acid, alkali solution, urea, calcium carbonate, ammonia and the like. [0037] When the microorganism is a transformant, and the recombinant DNA used for transforming the microorganism has an antibiotic-resistance gene, an antibiotic corresponding to the antibiotic-resistance gene possessed by the recombinant DNA may be added to the medium used for culturing the microorganism. When a culture of two or more kinds of microorganisms or a treated culture is used as the biocatalyst, respective microorganisms are cultured separately or in the same medium according to the above-mentioned method and the resulting product can be used. [0038] When two or more kinds of microorganisms are cultured in the same medium, these microorganisms may be cultured simultaneously, or one microorganism may be cultured and the rest of the microorganisms may be cultured in the same medium during the culture of the first microorganism or after 16 completion of the culture thereof. Examples of the treated culture of the microorganism include a surfactant-treated culture, organic solvent-treated culture or cytolytic enzyme-treated culture obtained by treating the culture of the microorganism obtained by the above-mentioned method with a surfactant, an organic solvent or a cytolytic enzyme such as lysozyme and the like. A surfactant, an organic solvent or a cytolytic enzyme may be used singly to treat the culture of the microorganism, or they may be combined to treat the culture of the microorganism. In addition, a concentrate or dried product of the culture of the microorganism, which is obtained by concentrating or drying, in a concentrating machine, a desiccator and the like, a culture of the microorganism obtained by the above-mentioned method, cells obtained by solid-liquid separation of the culture of the microorganism by filtration, centrifugation and the like, and the dried product of the cells obtained by drying the cells in a desiccator and the like can be mentioned. Furthermore, the surfactant-treated product, the organic solvent-treated product, the cytolytic enzyme-treated product and the like of the cells can be mentioned, which are obtained by treating the cells with a surfactant, an organic solvent or a cytolytic enzyme such as lysozyme and the like, or a combination of these treated products. [0039] When two or more kinds of microorganisms are used, two or more kinds of treated cultures may be individually used as biocatalysts having the CDP-choline-producing activity, or a mixture obtained by mixing these treated products of the cultures may be used as the biocatalyst having a CDP-choline- producing activity. CDP-choline can be produced by bringing the above- mentioned biocatalyst in contact with a precursor of UTP and choline or phosphorylcholine or a salt thereof in a medium to allow production and accumulation of CDP-choline in the medium 17 and recovering CDP-choline from the medium. [0040] Examples of the precursor of UTP include orotic acid, OMP, uracil, uridine, UMP, UDP and the like, with preference given to orotic acid and uracil. A specific method of producing and accumulating CDP- choline comprises mixing the above-mentioned biocatalyst and the precursor of UTP and choline or phosphorylcholine or a salt thereof in a medium, adding other components as necessary to the obtained mixture and maintaining the mixture at 20 - 50°C for 2-48 hours while keeping pH 5 - 11, more preferably 6 - 10. [0041] The amount of the biocatalyst to be used varies depending on the specific activity and the like of the biocatalyst. For example, when the culture of the microorganism or the treated product of the culture is used as the biocatalyst, it is preferably used in an amount of 5 - 500 mg, preferably 10 - 300 mg, relative to 1 mg of choline chloride, as wet cells obtained by centrifuging the culture or the treated culture. Examples of choline, phosphorylcholine and salts thereof include choline, choline halides such as choline chloride, choline bromide, choline iodide and the like, choline bicarbonate, choline methylsulfate, choline dihydrogen citrate, choline bitartrate, phosphorylcholine, phosphorylcholine halides such as phosphorylcholine chloride, etc. and the like, preferably choline or phosphorylcholine halide, more preferably choline chloride or phosphorylcholine chloride. [0042] The precursor of UTP, choline, phosphorylcholine and salts thereof may be obtained by chemical synthesis, or obtained from an organism by a fermentation method and the like. In addition, it is not necessarily to purify them completely. Furthermore, all of these substrates are commercially available and can be obtained easily. 18 The concentration of the precursor of UTP, choline, phosphorylcholine and salts thereof is preferably 1 itimol/L - 1 mol/L, more preferably 10 - 100 mmol/L. [0043] Examples of other necessary components include energy donor necessary for producing CDP-choline, phosphate ion, magnesium ion, ammonium ion, surfactant, organic solvent and the like. Addition of these components is not necessary when they are provided in necessary guantities from a biocatalyst and the like. As the energy donor, sugars such as glucose, fructose, sucrose and the like, molasses, starch hydrolysate and the like, and amino acids such as glycine, alanine and the like can be used. They are preferably used at the concentration of 0.02 - 2.0 mol/L. [0044] As the phosphate ion, inorganic phosphate such as orthophosphoric acid, pyrophosphoric acid, polyphosphoric acid such as tripolyphosphoric acid, tetrapolyphosphoric acid and the like, polymetaphosphoric acid, monopotassium phosphate, dipotassium phosphate, monosodium phosphate, disodium phosphate, etc. and the like can be used. These phosphate ions are preferably used at a concentration of 10 - 500 mmol/L. As the magnesium ion, inorganic magnesium salt such as magnesium sulfate, magnesium nitrate, magnesium chloride and the like, and organic magnesium salt such as magnesium citrate and the like can be used. Magnesium ion is preferably used at a concentration of 5 - 200 mmol/L. [0045] As the ammonium ion, aqueous ammonia, ammonia gas, various inorganic or organic ammonia salts, yeast extract, corn steep liquor and the like can be used. In addition, an organic nutrient source such as glutamine, peptide containing glutamine, casamino acid and the like can also be used in place of ammonium ion. The concentration of these ammonium 19 ions is preferably used at a concentration of 10 mmol/L - 2 mol/L. [0046] As the surfactant, an anionic surfactant such as sodium dioctylsulfosuccinate (e.g., Rapisol B-80, manufactured by NOF Corporation), lauroyl sarcosinate and the like, a non-ionic surfactant such as polyoxyethylene cetyl ether (e.g., Nonion P-208, manufactured by NOF Corporation) and the like, tertiary amines such as alkyldimethylamine (e.g., tertiary amine FB, manufactured by NOF Corporation), etc. and the like can also be used as long as production of CDP-choline is promoted. These are used in the range of generally 0.1 - 100 g/L, preferably 1-50 g/L. [0047] Examples of the organic solvent include xylene, toluene, aliphatic alcohol (methyl alcohol, ethyl alcohol, butyl alcohol, etc.), acetone, ethyl acetate, dimethyl sulfoxide and the like. These are used at a concentration of generally 0.1 - 100 mL/L, preferably 1-50 mL/L. As the medium in which the biocatalyst is contacted with the precursor of UTP and choline or phosphorylcholine or a salt thereof, the medium for culturing the microorganism to be used as a biocatalyst, the culture of the microorganism, the supernatant of the culture and the like can be used, and an aqueous medium can also be used. [0048] Examples of the aqueous medium include water and buffers such as phosphate buffer, HEPES (N-2-hydroxyethylpiperazine-N- ethanesulfonic acid) buffer, tris [tris(hydroxymethyl)aminomethane] hydrochloride buffer and the like. Any organic solvent may be added to the medium as long as the reaction is not inhibited. Examples of the organic solvent include acetone, ethyl acetate, dimethyl sulfoxide, xylene, methyl alcohol, ethyl alcohol, butanol and the like. 20 [0049] Examples of the method of producing CDP-choline includes a method (Japanese Patent No. 3369236, US Patent No. 6387667) including producing CDP-choline using Corynebacterium ammoniagenes ATCC21170 and Escherichia coli MM294/pCKG55 strain (FERM BP-3717) as the biocatalyst. Examples of the CDP-choline-producing enzyme include one or more enzymes selected from the group consisting of orotate phosphoribosyl transferase, orotidine-5'-monophosphate decarboxylase, uridine phosphorylase, uracil phosphoribosyltransferase, uridine kinase, uridylate and cytidylate kinases, nucleoside diphosphate kinase, PyrG, CCT and CKI. [0050] A crude enzyme or a purified enzyme obtained by disrupting the microorganism having the above-mentioned enzyme activity with a homogenizer and the like, and further applying a general enzyme purification method such as salting out, isoelectric point precipitation, organic solvent precipitation, dialysis, various chromatography treatments and the like can be used as the CDP-choline-producing enzyme. In addition, the disrupted product of the microorganism may be used as it is as the above-mentioned enzyme. [0051] In addition, the above-mentioned disrupted product of microorganism, crude enzyme or purified enzyme may be immobilized on a water-insoluble carrier, gel and the like, and used as the above-mentioned enzyme. CDP-choline can be produced by bringing the above- mentioned enzyme in contact with the precursor of UTP and choline or phosphorylcholine or a salt thereof in the medium to produce and accumulate CDP-choline, and recovering CDP- choline from the medium. [0052] A specific method of producing and accumulating CDP- 21 choline comprises mixing the above-mentioned enzyme and the precursor of UTP and choline or phosphorylcholine or a salt thereof in a medium, adding other components as necessary to the obtained mixture and maintaining the mixture at 20 - 50°C for 2-48 hours while keeping pH 5 - 11, more preferably 6 - 10. The amount of the CDP-producing enzyme to be used varies depending on the specific activity and the like of the enzyme. For example, when the crude enzyme is used as the enzyme, it is preferably used in an amount of 1 \iq - 500 mg, preferably 10 jig - 300 mg, relative to 1 mg of choline chloride. [0053] The precursor of UTP, choline, phosphorylcholine, a salt thereof to be added and other components to be added as necessary when producing and accumulating CDP-choline using the CDP-choline-producing enzyme are similar to those used for producing and accumulating CDP-choline using the culture and the like of the microorganism as mentioned above. Furthermore, adenosine-5'-triphosphate and the like may be added as necessary as an energy donor, and 5-phosphoribosyl diphosphate may be further added. [0054] As the medium in which the CDP-choline-producing enzyme is brought into contact with the precursor of UTP and choline or phosphorylcholine or a salt thereof, the medium used for culturing the microorganism to be used as the biocatalyst, the culture, the supernatant of the culture and the like of the microorganism may be used, or an aqueous medium may also be used. Examples of the aqueous medium include water, buffers such as phosphate buffer, HEPES (N-2-hydroxyethylpiperazine-N- ethanesulfonic acid) buffer, tris [tris(hydroxymethyl)aminomethane] hydrochloride and the like. [0055] For preparing the CDP-choline solution from the medium in 22 which CDP-choline has been produced and accumulated as mentioned above, a solid may be separated and removed from the medium according to a method using membrane separation, filtration, centrifugation and the like. Examples of the nucleic acid analogue contained in the CDP-choline solution prepared by the above-mentioned method include uracil, UTP and the like. CDP-choline and nucleic acid analogs can be analyzed by a conventional method using high performance liquid chromatography (UV detection). [0056] The present invention is explained in more detail in the following by referring to Examples, which are not to be construed as limitative. Example 1 Purification of CDP-choline using strongly acidic cation exchange resin CDP-choline (50 g; manufactured by Wako Pure Chemical Industries, Ltd.), uracil (2 g; manufactured by Nacalai Tesque) and UTP (1 g; manufactured by Nacalai Tesque) were dissolved in water to prepare 5 L of CDP-choline solution. The solution was adjusted to pH 3.0 with sulfuric acid and applied to a column packed with a strongly acidic cation exchange resin Diaion PK208 (H-type) (10 L) with a crosslinking degree of 4%. Subsequently, water was applied, and a fraction wherein each concentration of uracil and UTP was less than 0.1% (w/w) relative to CDP-choline was obtained as a result of analysis by high performance liquid chromatography. The CDP-choline fraction was concentrated to 100 mL, and ethanol (350 mL) was gradually added. The precipitated crystals were collected by filtration, washed with 100% ethanol solution, and dried under reduced pressure at 20°C for 3 days. As the result, 40 g of CDP-choline crystals, wherein each concentration of uracil and UTP was less than 0.1% (w/w) relative to CDP-choline, were obtained. 23 Example 2 Purification of CDP-choline from culture of microorganism having an activity to produce CDP-choline Escherichia coli MM294/pCKG55 strain (FERM BP-3717) having enzyme activities of PyrG, CCT and CKI was inoculated to a test tube containing L medium (10 mL) [liquid medium containing bactotripton (10 g/L; manufactured by Difco), yeast extract (5 g/L; manufactured by Difco) and NaCl (5 g/L) and adjusted to pH 7.2] supplemented with ampicillin (50 jig/mL) , and cultured with shaking at 25°C, 300 rpm for 24 hours. The culture thus obtained (20 mL) was inoculated to 2 L of conical flask with baffles containing L medium (400 mL) supplemented with ampicillin (50 \|j.g/mL) and cultured with rotary shaking at 25°C, 190 rpm for 16 hours. The culture thus obtained (125 mL) was inoculated to a 5 L culture bath containing a liquid medium (2.5 L; nonadjusted pH) with a composition of glucose (5 g/L; separately sterilized), peptone (5 g/L; manufactured by Kyokuto Pharmaceutical Industrial), Na2HP04 (6 g/L), KH2P04 (3 g/L), NaCl (5 g/L), NH4C1 (1 g/L), MgS04-7H20 (250 mg/L; separately sterilized) and vitamin Bl (4 [J.g/L; separately sterilized), and cultured with shaking at 600 rpm under culture condition of airflow 2.5 L/minute, at 25°C for 11 hours and then at 32°C for 13 hours, while adjusting the mixture to pH 7.0 with 14% aqueous ammonia. During culturing, a feed solution having a composition of glucose (167 g/L), peptone (167 g/L) was fed with a peristaltic pump at a rate of 30 mL/ hours during the period of from 11 hours to 24 hours from the start of the culturing. [0059] On the other hand, Corynebacterium*ammoniagenes ATCC21170 strain having an activity to produce UTP from orotic acid was inoculated to a test tube containing a liquid medium (10 mL) having a composition of glucose (50 g/L), polypeptone (10 g/L; manufactured by Daigo Eiyo Chemicals), yeast extract (10 g/L; manufactured by Daigo Eiyo Chemicals), urea (5 g/L), (NH4)2S04 24 (5 g/L), KH2P04 (1 g/L) , K2HP04 (3 g/L) , MgS04'7H20 (1 g/L) , CaCl2-2H20 (0.1 g/L), FeS04-7H20 (10 mg/L), ZnS04-7H20 (10 mg/L) , MnS04-4 - 6H20 (20 mg/L), L-cysteine (20 mg/L), calcium D- pantothenate (10 mg/L), vitamin Bl (5 mg/L), nicotinic acid (5 mg/L) and biotin (30 jig/L; adjusted to pH 7.2 with sodium hydroxide), and cultured with reciprocal shaking at 300 rpm at 28°C for 24 hours. The culture thus obtained (20 mL) was inoculated to a 2 L conical flask with baffle containing a liquid medium (230 mL) having the same composition as the above-mentioned, and cultured with rotary shaking at 190 rpm at 28°C for 24 hours. The culture thus obtained (250 mL) was inoculated to a 5 L culture bath containing a liquid medium (2.5 L) having a composition of glucose (100 g/L), meat extract (10 g/L), polypeptone (10 g/L), KH2P04 (1 g/L), K2HP04 (1 g/L), MgS04.7H20 (1 g/L), CaCl2-2H20 (0.1 g/L), FeS04-7H20 (20 mg/L), ZnS04-7H20 (10 mg/L), MnS04-4 - 6H20 (20 mg/L), p-alanine (15 mg/L), L-cysteine (20 mg/L), biotin (100 p,g/L) , urea (2 g/L; separately sterilized) and vitamin Bl (5 mg/L; separately sterilized) (adjusted to pH 7.2 with sodium hydroxide) and seed-cultured with shaking at 600 rpm at 32°C under culture condition of airflow 2.5 L/minute while adjusting the mixture to pH 6.8 with concentrated aqueous ammonia. At the time point when glucose in the supernatant of the above-mentioned seed- culture medium was consumed, the culture (350 mL) was aseptically collected, inoculated to a 5 L culture bath containing a liquid medium (2.5 L) having a composition of glucose (180 g/L), KH2P04 (10 g/L), K2HP04 (10 g/L), MgS04-7H20 (10 g/L), CaCl2-2H20 (0.1 g/L), FeS04-7H20 (20 mg/L), ZnS04-7H20 (10 mg/L), MnS04-4 - 6H20 (20 mg/L; separately sterilized), 0- alanine (15 mg/L), L-cysteine (20 mg/L), sodium glutamate (1 g/L) , biotin (100 \iq/L) , urea (2 g/L; separately sterilized) and vitamin Bl (5 mg/L; separately sterilized) (adjusted to pH 7.2 with sodium hydroxide) and main-cultured with shaking at 600 rpm at 32°C under culture condition of airflow 2.5 L/minute while adjusting the mixture to pH 6.8 with concentrated 25 aqueous ammonia. The culturing was ceased at the time point when glucose in the culture supernatant was consumed. [0060] The culture (360 mL) of Escherichia coli MM294/pCKG55 strain and the culture (360 mL) of Corynebacterium-ammoniagenes ATCC21170 strain thus obtained were placed in a 2 L culture bath, glucose (100 g/L), orotic acid (10 g/L), choline chloride (8.4 g/L), MgS04-7H20 (5 g/L) and xylene (20 mL/L) were added thereto, and distilled water was added to allow the total amount to be 800 mL. The mixture was reacted with shaking at 800 rpm at 32°C under the condition of airflow 0.8 L/minute while adjusting the mixture to pH 7.2 with ION sodium hydroxide. During the reaction, KH2P04 was added as appropriate to keep the concentration of phosphoric acid in the supernatant of the reaction mixture at 1 - 5 g/L as KH2P04. The reaction was carried out for 23 hours to obtain 11.0 g/L of CDP-choline. [0061] Four batches of the above-mentioned reaction mixture were adjusted to pH 1.0 with sulfuric acid, cells were separated by centrifugation (7000 rpm, 10 min), and water was added to the obtained supernatant to make the total amount 6 L (CDP-choline 6.0 g/L, uracil 0.5 g/L, UTP 1.0 g/L). This cytidinediphosphoric choline solution was applied to a column packed with a strongly acidic cation exchange resin Diaion SK104 (H-type, 10 L) having a crosslinking degree of 4%. Subsequently, water was applied, and a fraction, in which each concentration of uracil, UTP was less than 0.1%(w/w) relative to CDP-choline (analyzed by high performance liquid chromatography) was collected. The CDP-choline fraction was decolorized using activated carbon, and then concentrated to 100 mL. Ethanol (350 mL) was gradually added to the concentrated solution, and the precipitated crystals were collected by filtration. The obtained crystals were washed with 100% ethanol solution, and then dried under reduced 26 pressure at 20°C for 3 days. As a result, CDP-choline crystals (18 g), in which each concentration of uracil, UTP was less than 0.1% (w/w) relative to CDP-choline, were obtained. Example 3 Purification of CDP-choline sodium salt from a culture of microorganism having an ability to produce CDP-choline Four batches of the reaction mixture obtained in the same manner as in Example 2 were adjusted to pH 1.0 with sulfuric acid, cells were separated by centrifugation (7000 rpm, 10 min), and water was added to the obtained supernatant to make the total amount 6 L (CDP-choline 6.0 g/L, uracil 0.5 g/L, UTP 1.0 g/L). This cytidine diphosphate choline solution was applied to a column packed with a strongly acidic cation exchange resin Diaion SK104 (H-type, 10 L) having a crosslinking degree of 4%. Subsequently, water was applied, and a fraction, in which each concentration of uracil, UTP was less than 0.1%(w/w) relative to CDP-choline (analyzed by high performance liquid chromatography) was collected. The CDP- choline fraction was adjusted to pH 7.5 with sodium hydroxide, decolorized with activated carbon, and then concentrated to 100 mL. Ethanol (400 mL) was gradually added to the concentrated solution, and the precipitated crystals were collected by filtration. The obtained crystals were washed with 100% ethanol solution, and then dried under reduced pressure at 20°C for 3 days. As a result, CDP-choline sodium salt crystals (20 g), in which each concentration of uracil, UTP was less than 0.1% (w/w) relative to CDP-choline, were obtained. [0063] From the foregoing results, it has been clarified that CDP-choline or a salt thereof free from impurity can be obtained conveniently by treating a CDP-choline solution containing a nucleic acid analogue only once with a strongly acidic cation exchange resin. Industrial Applicability 27 [0064] According to the present invention, CDP-choline and a salt thereof are provided at a low cost. 28 Claims 1. A method of purifying cytidine diphosphate choline (hereinafter abbreviated as CDP-choline), which comprises contacting a CDP-choline solution containing a nucleic acid analogue and having a pH of not less than 0.5 and not more than 5.0 with an H-type strongly acidic cation exchange resin, and eluting CDP-choline adsorbed onto the resin with water or an aqueous solution having an ion concentration of not more than 0.1 mol/L to separate and purify CDP-choline. 2. The method of claim 1, wherein the CDP-choline solution is prepared from a resulting medium containing CDP-choline, which has been produced and accumulated therein by placing a biocatalyst having an activity to produce CDP-choline from a precursor of uridine-5'-triphosphate (hereinafter abbreviated as UTP) and choline or phosphorylcholine in an aqueous medium together with the precursor of UTP and choline or phosphorylcholine or a salt thereof. 3. The method of claim 2, wherein the biocatalyst comprises a culture or a treated culture of a microorganism capable of producing UTP from a precursor of UTP, and a culture or a treated culture of a microorganism capable of producing CDP- choline from UTP and choline or phosphorylcholine. 4. The method of claim 2, wherein the biocatalyst comprises an enzyme that catalyzes a reaction to produce CDP-choline from a precursor of UTP and choline or phosphorylcholine. 5. The method of claim 4, wherein the enzyme that catalyzes a reaction to produce CDP-choline is an enzyme selected from the group consisting of orotate phosphoribosyl transferase, orotidine-5'-monophosphate decarboxylase, uridine phosphorylase, uracil phosphoribosyltransferase, uridine 29 kinase, uridylate and cytidylate kinases, nucleoside diphosphate kinase, cytidine-5'-triphosphate synthase, choline phosphate cytidyltransferase and choline kinase. 30 6. The method of any of claims 1 to 5, wherein the nucleic acid analogue is selected from uracil and UTP. A method of purifying cytidine diphosphate choline, which comprises contacting a cytidine diphosphate choline solution containing a nucleic acid analogue and having a pH of not less than 0.5 and not more than 5.0 with an H-type strongly acidic cation exchange resin, and eluting cytidine diphosphate choline adsorbed onto the resin with water or an aqueous solution having an ion concentration of not more than 0.1 mol/L to separate and purify the cytidine diphosphate choline.

Full Text

DESCRIPTION
METHOD OP PURIFYING CYTIDINE DIPHOSPHATE CHOLINE
Technical Field
[0001]
The present invention relates to a method of purifying
cytidine diphosphate choline useful as a starting material of
pharmaceutical products and a starting material of nutritious
foods.
Background Art
[0002]
As the method of purifying cytidine diphosphate choline
(hereinafter to be abbreviated as CDP-choline), chemical
synthetic methods (patent reference 1, patent reference 2) and
methods using a culture of a microorganism or an enzyme
(patent reference 3, patent reference 4) are known. As the
method of purifying CDP-choline produced by a chemical
synthetic method, a method using an anion exchange resin
(patent reference 1) and a method using both of a strongly
acidic ion exchange resin and a weakly basic ion exchange
resin (patent reference 2) are known. In the latter method,
two kinds of ion exchange resins are used. Moreover, while
phosphorylcholine and cytidine-5'-monophosphate (hereinafter
to be abbreviated as CMP) contained in a CDP-choline solution
can be separated, uracil or uridine-5'-triphosphate
(hereinafter to be abbreviated as UTP) cannot be separated
efficiently by this method.
[patent reference l] Japanese Published Examined
Patent Application No. 6558/1988
[patent reference 2] Japanese Published Examined
Patent Application No.31306/1994
[patent reference 3] Japanese Patent No.3369236
[patent reference 4] WO99/49073
Disclosure of the Invention
Problems to be Solved by the Invention
[0003]
1

An object of the present invention is to provide a method
of purifying CDP-choline, in which method CDP-choline can be
separated from a nucleic acid analogue conveniently.
Means of Solving the Problems
[0004]
The present invention relates to the following (1) - (6).
(1) A method of purifying CDP-choline, which comprises
contacting a CDP-choline solution containing a nucleic acid
analogue and having a pH of not less than 0.5 and not more
than 5.0 with an H-type strongly acidic cation exchange resin,
and eluting CDP-choline adsorbed onto the resin with water or
an aqueous solution having an ion concentration of not more
than 0.1 mol/L to separate and purify CDP-choline.
(2) The method of the above-mentioned (1), wherein the CDP-
choline solution is prepared from a resulting medium
containing CDP-choline, which has been produced and
accumulated therein by placing a biocatalyst having an
activity to produce CDP-choline from a precursor of UTP and
choline or phosphorylcholine in an aqueous medium together
with the precursor of UTP and choline or phosphorylcholine or
a salt thereof.

(3) The method of the above-mentioned (2), wherein the
biocatalyst comprises a culture or a treated culture of a
microorganism capable of producing UTP from a precursor of UTP,
and a culture or a treated culture of a microorganism capable
of producing CDP-choline from UTP and choline or
phosphorylcholine.
(4) The method of the above-mentioned (2), wherein the
biocatalyst comprises an enzyme that catalyzes a reaction to
produce CDP-choline from a precursor of UTP and choline or
phosphorylcholine.
(5) The method of the above-mentioned (4), wherein the enzyme
that catalyzes a reaction to produce CDP-choline is an enzyme
selected from the group consisting of orotate phosphoribosyl
transferase, orotidine-5'-monophosphate decarboxylase, uridine
2

phosphorylase, uracil phosphoribosyltransferase, uridine
kinase, uridylate and cytidylate kinases, nucleoside
diphosphate kinase, cytidine-5'-triphosphate synthase, choline
phosphate cytidyltransferase and choline kinase.
(6) The method of any of the above-mentioned (1) to (5),
wherein the nucleic acid analogue is selected from uracil and
UTP.
Effect of the Invention
[0005]
The present invention provides CDP-choline and a salt
thereof at a low cost.
Best Mode for Carrying Out the Invention
[0006]
The CDP-choline solution to be used in the present
invention may be prepared by any method as long as it contains
a nucleic acid analogue and has a pH of not less than 0.5 and
not more than 5.0. When the prepared CDP-choline solution has
a pH of 0.5 - 5.0, it is directly used. When the pH is higher
than 5.0, an acid such as hydrochloric acid, sulfuric acid,
nitric acid, phosphoric acid and the like, is added, and when
the pH is lower than 0.5, an alkali such as sodium hydroxide,
potassium hydroxide and the like is added to adjust to pH 0.5
- 5.0, preferably 1.0 - 3.5, before use.
[0007]
The H-type strongly acidic cation exchange resin may be
of a gel type or a porous type, as long as it is an H-type
strongly acidic cation exchange resin. Specific examples
thereof include the Dowex series (e.g., HCR-S, HCR-W2,
Marathon C, Monosphere 650C, MSC-1, Monosphere 88, 50W*2,
50W*4, 50W*8 etc.) manufactured by Dow Chemical Company,
Diaion SK series (e.g., SK1B, SK104, SK110, SK112 etc.)
manufactured by Mitsubishi chemical, Diaion PK series (e.g.,
PK208, PK212, PK220, PK228 etc.) manufactured by Mitsubishi
Chemical, Amberlite series (e.g., IR120B, IR124 etc.)
manufactured by Rohm and Haas and the like.
3

[0008]
Degree of crosslinking of the H-type strongly acidic
cation exchange resin is not particularly limited as long as
CDP-choline and nucleic acid analogue can be separated at the
degree of crosslinking. It is preferably 2 - 10%, more
preferably 4-6%.
In the present invention, the H-type strongly acidic
cation exchange resin is preferably used in the form of a
packed column, and as the column to be used in the present
invention, any column may be used.
The CDP-choline solution containing a nucleic acid
analogue and having a pH of not less than 0.5 and not more
than 5.0 is contacted with the H-type strongly acidic cation
exchange resin by applying the solution to a column packed
with the resin and the like to allow adsorption of CDP-choline
onto the resin. For example, when the solution is applied to a
column packed with an H-type strongly acidic cation exchange
resin having a degree of crosslinking of 2 - 10%, the flow
rate (SV) is 0.1 - 5.0, preferably 0.2 - 3.0.
[0009]
By the above-mentioned adsorption treatment, a nucleic
acid analogue contained in the solution, particularly a
pyrimidine type nucleic acid substance such as orotic acid,
orotidine-5'-monophosphate (hereinafter to be abbreviated as
OMP), uridine-5'-monophosphate (hereinafter to be abbreviated
as UMP), uridine-5'-diphosphate (hereinafter to be abbreviated
as UDP), UTP, CMP, cytidine-5'-diphosphate (hereinafter to be
abbreviated as CDP), cytidine-5'-triphosphate (hereinafter to
be abbreviated as CTP) and the like can be efficiently
separated or removed from CDP-choline. Out of the nucleic acid
analogues, uracil and UTP are particularly preferably
separated and removed, since they hardly adsorb to or do not
at all adsorb to the resin, when a CDP-choline solution
containing uracil and UTP is brought into contact with the
resin at a pH of not less than 0.5 and not more than 5.0.
4

[0010]
CDP-choline adsorbed on the H-type strongly acidic cation
exchange resin is eluted by applying an aqueous solution
having an ion concentration of not more than 0.1 mol/L,
preferably not more than 0.03 mol/L, more preferably water,
and the like, whereby CDP-choline is eluted from the resin,
for the separation and purification.
Where necessary, the CDP-choline-containing solution
collected by the above-mentioned elution step may be subjected
to an activated carbon treatment or decolorization treatment
using a nonpolar porous synthetic adsorbent, for example,
Diaion HP series (e.g., HP20, HP21 etc.) manufactured by
Mitsubishi Chemical, Diaion SP800 series (e.g., SP825, SP850
etc.) manufactured by Mitsubishi Chemical, Diaion SP200 series
(e.g., SP207 etc.) manufactured by Mitsubishi Chemical,
Amberlite XAD series (e.g., XAD4, XAD7HP, XAD16HP, XAD1180,
XAD2000 etc.) manufactured by Rohm and Haas and the like.
[0011]
The above-mentioned CDP-choline-containing solution or
decolorized solution is adjusted to pH 2.0 - 4.0 with acid or
alkali. After concentration is carried out as necessary, the
concentration of CDP-choline is adjusted to 50 - 800 g/L,
preferably 100 - 700 g/L, and crystals of CDP-choline can be
obtained using an organic solvent, preferably a hydrophilic
organic solvent such as acetone, ethanol, methanol, propanol
and the like.
In addition, the above-mentioned CDP-choline-containing
solution or decolorized solution is adjusted to pH 5.0 - 9.5
with sodium hydroxide. After concentration is carried out as
necessary, the concentration of CDP-choline is adjusted to 50
- 800 g/L, preferably 100 - 700 g/L, and crystals of CDP-
choline sodium salt can be obtained using an organic solvent,
preferably a hydrophilic organic solvent such as acetone,
ethanol, methanol, propanol and the like.
[0012]
5

Examples of the method of obtaining CDP-choline crystals
using an organic solvent include a method comprising adding an
organic solvent to a CDP-choline solution to allow
precipitation of crystals, and a method comprising adding
dropwise a CDP-choline solution to a large amount of an
organic solvent to allow precipitation of crystals.
In the present invention, the CDP-choline solution
containing a nucleic acid analogue may be any as long as it is
a solution containing CDP-choline and a nucleic acid analogue.
Examples thereof include a solution produced by a chemical
synthetic method, a method using a biocatalyst having an
activity to produce CDP-choline from a precursor of UTP and
choline or phosphorylcholine (hereinafter to be abbreviated as
CDP-choline-producing activity).
[0013]
Examples of the biocatalyst include a culture of a
microorganism having a CDP-choline-producing activity, a
treated culture thereof, an enzyme that catalyzes a reaction
to produce CDP-choline and the like.
As the microorganism, any microorganism can be used as
long as it has a CDP-choline-producing activity. A
microorganism inherently having a CDP-choline-producing
activity can be used as it is for the production of CDP-
choline, and a microorganism naturally having no CDP-choline-
producing activity can be used for the production of CDP-
choline by introducing a DNA encoding an enzyme that catalyzes
a reaction to produce CDP-choline from a precursor of UTP and
choline or phosphocholine. Preferable examples of the
microorganism include microorganisms belonging to the genera
Escherichia, Serratia, Bacillus, Brevibacterium,
Corynebacterium, Microbacterium, Pseudomonas, Streptococcus,
Sinorhizobium, Haemophilus, Arthrobacter, Aureobacterium,
Cellulomonas, Clavibacter, Curtobacterium, Pimerobacter,
Saccharomyces, Schizosaccharomyces, Kluyveromyces,
Trichosporon, Schwanniomyces, Pichia, Candida and the like.
6

[0014]
Examples of the microorganism belonging to the genus
Escherichia include microorganisms belonging to Escherichia
coli such as Escherichia coli MM294, Escherichia coli XLl-Blue,
Escherichia coli XL2~Blue, Escherichia coli DH1, Escherichia
coli MC1000, Escherichia coli KY3276, Escherichia coli W1485,
Escherichia coli JM109, Escherichia coli HB101, Escherichia
coli No.49, Escherichia coli W3110, Escherichia coli NY49,
Escherichia coli GI698, Escherichia coli TBI and the like.
Examples of the microorganism belonging to the genus Serratia
include Serratia ficaria, Serratia fonticola, Serratia
liquefaciens, Serratia marcescens and the like. Examples of
the microorganism belonging to the genus Bacillus include
Bacillus subtilis, Bacillus megaterium, Bacillus
amyloliquefaciens and the like. Examples of the microorganism
belonging to the genus Brevibacterium include Brevibacterium
immariophilum, Brevibacterium saccharolyticum, Brevibacterium
flavum, Brevibacterium lactofermentum and the like. Examples
of the microorganism belonging to the genus Corynebacterium
include microorganisms belonging to Corynebacterium glutamicum
such as Corynebacterium glutamicum ATCC13032, Corynebacterium
glutamicum ATCC138 69 and the like, microorganisms belonging to
Corynebacterium ammoniagenes such as Corynebacterium
ammoniagenes ATCC6872, Corynebacterium ammoniagenes ATCC21170
and the like, microorganisms belonging to Corynebacterium
acetoacidophilum such as Corynebacterium acetoacidophilum
ATCC1387 0, etc. and the like. Examples of the microorganism
belonging to the genus Microbacterium include microorganisms
belonging to Microbacterium ammoniaphilum such as
Microbacterium ammoniaphilum ATCC15354 and the like,
Microbacterium lactium, Microbacterium imperiale and the like.
Examples of the microorganism belonging to the genus
Pseudomonas include Pseudomonas putida and the like. Examples
of the microorganism belonging to the genus Streptococcus
include Streptococcus pneumoniae and the like. Examples of the
7

microorganism belonging to the genus Sinorhizobium include
Sinorhizobium meliloti and the like. Examples of the
microorganism belonging to the genus Haemophilus include
Haemophilus influenzae and the like. Examples of the
microorganism belonging to the genus Arthrobacter include
Arthrobacter citreus, Arthrobacter globiformis and the like.
Examples of the microorganism belonging to the genus
Aureobacterium include Aureobacterium flavescens,
Aureobacterium saperdae, Aureobacterium testaceum and the like.
Examples of the microorganism belonging to the genus
Cellulomonas include Cellulomonas flavigena, Cellulomonas
carta and the like. Examples of the microorganism belonging to
the genus Clavibacter include Clavibacter michiganensis,
Clavibacter rathayi and the like. Examples of the
microorganism belonging to the genus Curtobacterium include
Curtobacterium albidum, Curtobacterium citreum, Curtobacterium
luteum and the like. Examples of the microorganism belonging
to the genus Pimerobacter include Pimerobacter simplex and the
like.
[0015]
Examples of the microorganism belonging to the genus
Saccharomyces include Saccharomyces cerevisiae and the like.
Examples of the microorganism belonging to the genus
Schizosaccharomyces include Schizosaccharomyces pombe and the
like. Examples of the microorganism belonging to the genus
Kluyveromyces include Kluyveromyces lactis and the like.
Examples of the microorganism belonging to the genus
Trichosporon include Trichosporon pullulans and the like.
Examples of the microorganism belonging to the genus
Schwanniomyces include Schwanniomyces alluvius and the like.
Examples of the microorganism belonging to the genus Pichia
include Pichia pastoris and the like. Examples of the
microorganism belonging to the genus Candida include Candida
utilis and the like.
[0016]
8

In addition, more preferable examples of the
microorganism include microorganisms belonging to the genera
Escherichia, Bacillus, Brevibacteriuirt, Corynebacterium and
Saccharomyces, further preferably, microorganisms belonging to
the genera Escherichia, Brevibacterium, Corynebacterium.
[0017]
Of the above-mentioned microorganisms, when a
microorganism naturally having a CDP-choline-producing
activity has only an insufficient CDP-choline-producing
activity, a recombinant DNA having a DNA encoding an enzyme
that catalyzes a reaction to produce CDP-choline from a
precursor of UTP and choline or phosphorylcholine may be
introduced into the microorganism according to a conventional
method, or a cell of different microorganism having the
activity may be fused therewith to prepare a microorganism
having an enhanced activity.
[0018]
As the microorganism having an enhanced activity or a
microorganism imparted with the activity, a transformant
obtained by introducing a DNA encoding an enzyme that
catalyzes the reaction into a microorganism according to the
following method can be preferably used.
Examples of the DNA encoding an enzyme that catalyzes a
reaction to produce CDP-choline from a precursor of UTP and
choline or phosphorylcholine (hereinafter to be abbreviated as
a CDP-choline-producing enzyme) include DNAs encoding orotate
phosphoribosyl transferase [EC 2.4.2.10] having an activity to
produce OMP from orotic acid, orotidine-5'-monophosphate
decarboxylase [EC 4.1.1.23] having an activity to produce UMP
from OMP, uridine phosphorylase [EC 2.4.2.3] having an
activity to produce uridine from uracil, uracil
phosphoribosyltransferase [EC 2.4.2.9] having an activity to
produce UMP from uracil, uridine kinase [EC 2.7.1.48] having
an activity to produce UMP from uridine, uridylate and
cytidylate kinases [EC 2.7.1.48] having an activity to produce
9

UDP from UMP, nucleoside diphosphate kinase [EC 2.7.4.6]
having an activity to produce UTP from UDP, cytidine-5'-
triphosphate synthetase [EC 6.3.4.2] (hereinafter to be
abbreviated as PyrG) having an activity to produce CTP from
UTP, choline kinase [EC 2.7.1.32] (hereinafter to be
abbreviated as CKI) having an activity to produce
phosphorylcholine from choline and choline phosphate
cytidyltransferase [EC 2.7.7.15] (hereinafter to be
abbreviated as CCT) having an activity to produce CDP-choline
from CTP and phosphorylcholine and the like.
[0019]
Preferable examples of the DNA encoding a CDP-choline-
producing enzyme include DNAs encoding PyrG, CKI and CCT.
A DNA encoding PyrG has been cloned from the chromosome
of Escherichia coli, and its entire nucleotide sequence has
been determined [J. Biol. Chem., 261, 5568(1986)]. Examples of
a recombinant having a DNA encoding PyrG include pMW6 [Biosci.
Biotechnol. Biochem., _6_1, 956 (1997)], which is a plasmid
having a 2426 bp NruI-PstI fragment containing a DNA encoding
PyrG derived from Escherichia coli inserted into an Smal-PstI
site of a multicloning site of vector pUC8 of Escherichia coli
[Gene, 19, 259 (1982)] and the like.
[0020]
The entire nucleotide sequence of the DNA encoding CCT
has been determined [Eur. J. Biochem., 169, 477(1987)].
Examples of a recombinant DNA having a DNA encoding CCT
include plasmid pCC41 [Biochemical, 1296 bp Dral fragment containing a DNA encoding CCT derived
from yeast inserted into a Smal site of a multicloning site of
vector pUC18 of Escherichia coli [Gene, 33, 103(1985)] and the
like.
[0021]
A DNA encoding CKI has also been cloned from a chromosome
of yeast in the same manner, and the entire nucleotide
sequence thereof has been determined [J. Biol. Chem., 264,
10

2053(1989)]. Examples of a recombinant DNA having a DNA
encoding CKI include plasmid pCKID [J. Biol. Chem., 264,
2053(1989)] having a 2692 bp Pstl-Hindlll fragment containing
a DNA encoding CKI derived from yeast inserted into a shuttle
vector YEpM4 of yeast and Escherichia coli, [Mol. Cell. Biol.,
1_, 29 (1987)] and the like.
[0022]
The above-mentioned plasmids can be isolated and purified
from Escherichia coli having these plasmids, according to a
known method [Nuc. Acids Res., 1_, 1513(1979)].
A biocatalyst having a CDP-choline-producing activity can
be obtained by obtaining a DNA encoding a CDP-choline-
producing enzyme from the plasmid obtained as mentioned above
according to, for example, Molecular Cloning, A Laboratory
Manual, Third Edition, Sambrook et al. edit, Cold Spring
Harbor Laboratory (2001), incorporating the DNA into an
expression vector to prepare a recombinant DNA, and
transforming the above-mentioned microorganism as a host cell.
[0023]
First, a DNA encoding PyrG, CCT or CKI is obtained from
the above-mentioned plasmid pMW6, plasmid pCC41 or plasmid
pCKID and, based on the obtained DNA, a DNA fragment having a
suitable length and containing a part encoding the polypeptide
is prepared as necessary.
Where necessary, moreover, a DNA wherein nucleotide of
the nucleotide sequence of a partial DNA fragment
corresponding to a CDP-choline-producing enzyme are
substituted so as to provide a codon optimal for the
expression of a host cell is prepared. The DNA is useful for
an efficient production of a CDP-choline-producing enzyme.
[0024]
A recombinant vector is produced by inserting the DNA
fragment or full-length DNA into the downstream of a promoter
of a suitable expression vector. In this case, DNA encoding a
CDP-choline-producing enzyme may be independently inserted
11

into an expression vector, or plural DNAs may be inserted into
the same expression vector.
The recombinant vector is introduced into a host cell
suitable for the expression vector.
[0025]
As the host cell, the above-mentioned microorganisms can
be mentioned.
As the expression vector, one capable of autonomous
replication or one capable of being incorporated into a
chromosome in the host cell, and having a promoter at a site
permitting transcription of a DNA encoding a CDP-choline-
producing enzyme can be used.
When prokaryote such as bacterium and the like is used as
a host cell, a recombinant vector having a DNA encoding a CDP-
choline-producing enzyme is preferably autonomously
replicatable in a prokaryote, as well as constituted with a
promoter, a ribosome binding sequence, the DNA and a
transcription termination sequence. A gene regulating the
promoter may also be contained.
[0026]
Examples of the expression vector include pBTrp2, pBTacl,
pBTac2 (all commercially available from Boehringer Mannheim),
pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by
Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8
(manufactured by QIAGEN), pKYPlO (Japanese Published
Unexamined Patent Application No. 110600/58), pKYP200 [Agric.
Biol. Chem., 4£, 669 (1984)], pLSAl [Agric. Biol. Chem. , 5_3,
277 (1989)], pGELl [Proc. Natl. Acad. Sci. USA, 82_, 4306
(1985)], pBluescript II SK(-) (manufactured by Stratagene),
pTrs30 [prepared from Escherichia coli JM109/pTrS30 (FERM BP-
5407)], pTrs32 [prepared from Escherichia coli JM109/pTrS32
(FERM BP-5408)], pGHA2 [prepared from Escherichia coli IGHA2
(FERM BP-400), Japanese Published Unexamined Patent
Application No. 221091/60], pGKA2 [prepared from Escherichia
coli IGKA2 (FERM BP-6798), Japanese Published Unexamined
12

Patent Application No. 221091/60], pTerm2 (US Patent 4686191,
US Patent 4939094, US Patent 5160735), pSupex, pUBHO, pTP5,
pCl94, pEG400 [J. Bacterid., 172, 2392 (1990)], pGEX
(manufactured by Pharmacia), pET system (manufactured by
Novagen) and the like.
[0027]
As the promoter, any promoter can be used as long as it
is functionable in the host cell. Examples of the promoter
include those derived from Escherichia coli, phage and the
like, such as trp promoter (PtrP), lac promoter,' PL promoter, PR
promoter, T7 promoter and the like. In addition, a promoter
having an artificially altered design such as a promoter
(Ptrp*2) in which two Ptrp are sequently connected in tandem, tac
promoter, lac T7 promoter and let I promoter, and the like can
also be used.
[0028]
It is preferable to use a plasmid in which the distance
between a Shine-Dalgarno sequence (ribosome binding sequence)
and an initiation codon is adjusted to a suitable distance
(e.g., 6 to 18 bases).
In the recombinant vector of the present invention, a
transcription termination sequence is not always necessarily
for the expression of a DNA encoding a CDP-choline-producing
enzyme. However, it is preferable to place a transcription
termination sequence directly downstream the structure gene.
[0029]
As a method of introducing the recombinant vector, any
method can be used as long as a DNA can be introduced into the
above-mentioned host cell. For example, a method using a
calcium ion [Proc. Natl. Acad. Sci. USA, 659, 2110 (1972)], a
protoplast method (Japanese Published Unexamined Patent
Application No. 248394/1988), the method described in Gene, 11_,
107 (1982) or Molecular & General Genetics, 1_68, 111 (1979)
and the like can be used.
[0030]
13

When a yeast is used as the host cell, for example, YEpl3
(ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19,
pHS15 and the like can be used as the expression vector.
As the promoter, any promoter can be used as long as it
permits expression in a yeast strain. Examples of the promoter
include promoters of genes of the glycolytic system such as
hexose kinase and the like, PH05 promoter, PGK promoter, GAP
promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat
shock polypeptide promoter, MFal promoter, CUP 1 promoter and
the like.
[0031]
As the method of introducing the recombinant vector, any
method can be used as long as it introduces the DNA into a
yeast. Examples thereof include an electroporation method
[Methods Enzymol., 1_94, 182 (1990)], a spheroplast method
[Proc. Natl. Acad. Sci. USA, 75_, 1929 (1978)], a lithium
acetate method [J. Bacteriology, 153, 163 (1983)], the method
described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and
the like.
[0032]
When the microorganism has only a part of the CDP-
choline-producing activity, two or more kinds of
microorganisms may be combined as appropriate to afford a CDP-
choline-producing activity, and used as the biocatalyst having
a CDP-choline-producing activity. Even when the microorganism
has a CDP-choline-producing activity, two or more kinds of
microorganisms can be combined.
Two or more kinds of microorganisms to be combined may be
selected from any of the above-mentioned microorganisms.
Examples of the combination include a combination of
microorganisms belonging to the same genus or different genera
selected from the microorganisms belonging to the genera
Escherichia, Serratia, Bacillus, Brevibacterium,
Corynebacterium, Microbacterium, Pseudomonas, Streptococcus,
Sinorhizobium, Haemophilus, Arthrobacter, Aureobacterium,
14

Cellulomonas, Clavibacter, Curtobacterium, Pimerobacter,
Saccharomyces, Schizosaccharomyces, Kluyveromyces,
Trichosporon, Schwanniomyces, Pichia and Candida, and the like.
[0033]
For example, a combination of the microorganism belonging
to the genus Corynebacterium and a microorganism belonging to
the genus Escherichia and the like can be mentioned. Specific
example is a combination of Corynebacterium ammoniagenes
ATCC21170 and Escherichia coli MM294/pCKG55 strain (FERM BP-
3717) (Japanese Patent No. 3369236, US Patent No. 6387667) and
the like.
The example of the culture of a microorganism having a
CDP-choline-producing activity, which is one of the
biocatalysts having a CDP-choline-producing activity, is a
culture obtained by culturing, according to a conventional
method, the microorganism obtained by the above-mentioned
method.
[0034]
When the microorganism is a prokaryote such as bacteria
and the like or a eukaryote such as yeast and the like, any of
a natural medium and a synthetic medium can be used as the
medium used for culturing the microorganism as long as it
contains a carbon source, a nitrogen source, inorganic salts
and the like to be assimilated by the microorganism, and
enables efficient culturing of the microorganism.
Any carbon source may be used as long as the
microorganism can assimilate it, and glucose, fructose,
sucrose, molasses containing them, carbohydrates such as
starch and starch hydrolysate, organic acid such as acetic
acid, propionic acid and the like, alcohols such as ethanol,
propanol and the like, and the like can be used.
[0035]
As the nitrogen source, ammonium salts of inorganic acid
or organic acid such as ammonia, ammonium chloride, ammonium
sulfate, ammonium acetate, ammonium phosphate and the like,
15

other nitrogen-containing compounds, peptone, meat extract,
yeast extract, corn steep liquor, casein hydrolysate, soybean
cake and soybean cake hydrolysate, various bacterial cells
obtained by fermentation and digests thereof, and the like can
be used.
[0036]
As the inorganic salt, monopotassium phosphate,
dipotassium phosphate, magnesium phosphate, magnesium sulfate,
sodium chloride, ferrous sulfate, manganese sulfate, copper
sulfate, calcium carbonate and the like can be used.
Culturing is performed under aerobic conditions by, for
example, shaking culturing, aerobic stirred culturing and the
like. The culturing temperature is preferably 15°C - 40°C and
the culturing time is generally 16 hours - 7 days. The pH is
preferably maintained 3-9 during culturing. The pH is
adjusted with inorganic or organic acid, alkali solution, urea,
calcium carbonate, ammonia and the like.
[0037]
When the microorganism is a transformant, and the
recombinant DNA used for transforming the microorganism has an
antibiotic-resistance gene, an antibiotic corresponding to the
antibiotic-resistance gene possessed by the recombinant DNA
may be added to the medium used for culturing the
microorganism.
When a culture of two or more kinds of microorganisms or
a treated culture is used as the biocatalyst, respective
microorganisms are cultured separately or in the same medium
according to the above-mentioned method and the resulting
product can be used.
[0038]
When two or more kinds of microorganisms are cultured in
the same medium, these microorganisms may be cultured
simultaneously, or one microorganism may be cultured and the
rest of the microorganisms may be cultured in the same medium
during the culture of the first microorganism or after
16

completion of the culture thereof.
Examples of the treated culture of the microorganism
include a surfactant-treated culture, organic solvent-treated
culture or cytolytic enzyme-treated culture obtained by
treating the culture of the microorganism obtained by the
above-mentioned method with a surfactant, an organic solvent
or a cytolytic enzyme such as lysozyme and the like. A
surfactant, an organic solvent or a cytolytic enzyme may be
used singly to treat the culture of the microorganism, or they
may be combined to treat the culture of the microorganism. In
addition, a concentrate or dried product of the culture of the
microorganism, which is obtained by concentrating or drying,
in a concentrating machine, a desiccator and the like, a
culture of the microorganism obtained by the above-mentioned
method, cells obtained by solid-liquid separation of the
culture of the microorganism by filtration, centrifugation and
the like, and the dried product of the cells obtained by
drying the cells in a desiccator and the like can be mentioned.
Furthermore, the surfactant-treated product, the organic
solvent-treated product, the cytolytic enzyme-treated product
and the like of the cells can be mentioned, which are obtained
by treating the cells with a surfactant, an organic solvent or
a cytolytic enzyme such as lysozyme and the like, or a
combination of these treated products.
[0039]
When two or more kinds of microorganisms are used, two or
more kinds of treated cultures may be individually used as
biocatalysts having the CDP-choline-producing activity, or a
mixture obtained by mixing these treated products of the
cultures may be used as the biocatalyst having a CDP-choline-
producing activity.
CDP-choline can be produced by bringing the above-
mentioned biocatalyst in contact with a precursor of UTP and
choline or phosphorylcholine or a salt thereof in a medium to
allow production and accumulation of CDP-choline in the medium
17

and recovering CDP-choline from the medium.
[0040]
Examples of the precursor of UTP include orotic acid, OMP,
uracil, uridine, UMP, UDP and the like, with preference given
to orotic acid and uracil.
A specific method of producing and accumulating CDP-
choline comprises mixing the above-mentioned biocatalyst and
the precursor of UTP and choline or phosphorylcholine or a
salt thereof in a medium, adding other components as necessary
to the obtained mixture and maintaining the mixture at 20 -
50°C for 2-48 hours while keeping pH 5 - 11, more preferably
6 - 10.
[0041]
The amount of the biocatalyst to be used varies depending
on the specific activity and the like of the biocatalyst. For
example, when the culture of the microorganism or the treated
product of the culture is used as the biocatalyst, it is
preferably used in an amount of 5 - 500 mg, preferably 10 -
300 mg, relative to 1 mg of choline chloride, as wet cells
obtained by centrifuging the culture or the treated culture.
Examples of choline, phosphorylcholine and salts thereof
include choline, choline halides such as choline chloride,
choline bromide, choline iodide and the like, choline
bicarbonate, choline methylsulfate, choline dihydrogen citrate,
choline bitartrate, phosphorylcholine, phosphorylcholine
halides such as phosphorylcholine chloride, etc. and the like,
preferably choline or phosphorylcholine halide, more
preferably choline chloride or phosphorylcholine chloride.
[0042]
The precursor of UTP, choline, phosphorylcholine and
salts thereof may be obtained by chemical synthesis, or
obtained from an organism by a fermentation method and the
like. In addition, it is not necessarily to purify them
completely. Furthermore, all of these substrates are
commercially available and can be obtained easily.
18

The concentration of the precursor of UTP, choline,
phosphorylcholine and salts thereof is preferably 1 itimol/L - 1
mol/L, more preferably 10 - 100 mmol/L.
[0043]
Examples of other necessary components include energy
donor necessary for producing CDP-choline, phosphate ion,
magnesium ion, ammonium ion, surfactant, organic solvent and
the like. Addition of these components is not necessary when
they are provided in necessary guantities from a biocatalyst
and the like.
As the energy donor, sugars such as glucose, fructose,
sucrose and the like, molasses, starch hydrolysate and the
like, and amino acids such as glycine, alanine and the like
can be used. They are preferably used at the concentration of
0.02 - 2.0 mol/L.
[0044]
As the phosphate ion, inorganic phosphate such as
orthophosphoric acid, pyrophosphoric acid, polyphosphoric acid
such as tripolyphosphoric acid, tetrapolyphosphoric acid and
the like, polymetaphosphoric acid, monopotassium phosphate,
dipotassium phosphate, monosodium phosphate, disodium
phosphate, etc. and the like can be used. These phosphate ions
are preferably used at a concentration of 10 - 500 mmol/L.
As the magnesium ion, inorganic magnesium salt such as
magnesium sulfate, magnesium nitrate, magnesium chloride and
the like, and organic magnesium salt such as magnesium citrate
and the like can be used. Magnesium ion is preferably used at
a concentration of 5 - 200 mmol/L.
[0045]
As the ammonium ion, aqueous ammonia, ammonia gas,
various inorganic or organic ammonia salts, yeast extract,
corn steep liquor and the like can be used. In addition, an
organic nutrient source such as glutamine, peptide containing
glutamine, casamino acid and the like can also be used in
place of ammonium ion. The concentration of these ammonium
19

ions is preferably used at a concentration of 10 mmol/L - 2
mol/L.
[0046]
As the surfactant, an anionic surfactant such as sodium
dioctylsulfosuccinate (e.g., Rapisol B-80, manufactured by NOF
Corporation), lauroyl sarcosinate and the like, a non-ionic
surfactant such as polyoxyethylene cetyl ether (e.g., Nonion
P-208, manufactured by NOF Corporation) and the like, tertiary
amines such as alkyldimethylamine (e.g., tertiary amine FB,
manufactured by NOF Corporation), etc. and the like can also
be used as long as production of CDP-choline is promoted.
These are used in the range of generally 0.1 - 100 g/L,
preferably 1-50 g/L.
[0047]
Examples of the organic solvent include xylene, toluene,
aliphatic alcohol (methyl alcohol, ethyl alcohol, butyl
alcohol, etc.), acetone, ethyl acetate, dimethyl sulfoxide and
the like. These are used at a concentration of generally 0.1 -
100 mL/L, preferably 1-50 mL/L.
As the medium in which the biocatalyst is contacted with
the precursor of UTP and choline or phosphorylcholine or a
salt thereof, the medium for culturing the microorganism to be
used as a biocatalyst, the culture of the microorganism, the
supernatant of the culture and the like can be used, and an
aqueous medium can also be used.
[0048]
Examples of the aqueous medium include water and buffers
such as phosphate buffer, HEPES (N-2-hydroxyethylpiperazine-N-
ethanesulfonic acid) buffer, tris
[tris(hydroxymethyl)aminomethane] hydrochloride buffer and the
like.
Any organic solvent may be added to the medium as long as
the reaction is not inhibited. Examples of the organic solvent
include acetone, ethyl acetate, dimethyl sulfoxide, xylene,
methyl alcohol, ethyl alcohol, butanol and the like.
20

[0049]
Examples of the method of producing CDP-choline includes
a method (Japanese Patent No. 3369236, US Patent No. 6387667)
including producing CDP-choline using Corynebacterium
ammoniagenes ATCC21170 and Escherichia coli MM294/pCKG55
strain (FERM BP-3717) as the biocatalyst.
Examples of the CDP-choline-producing enzyme include one
or more enzymes selected from the group consisting of orotate
phosphoribosyl transferase, orotidine-5'-monophosphate
decarboxylase, uridine phosphorylase, uracil
phosphoribosyltransferase, uridine kinase, uridylate and
cytidylate kinases, nucleoside diphosphate kinase, PyrG, CCT
and CKI.
[0050]
A crude enzyme or a purified enzyme obtained by
disrupting the microorganism having the above-mentioned enzyme
activity with a homogenizer and the like, and further applying
a general enzyme purification method such as salting out,
isoelectric point precipitation, organic solvent precipitation,
dialysis, various chromatography treatments and the like can
be used as the CDP-choline-producing enzyme. In addition, the
disrupted product of the microorganism may be used as it is as
the above-mentioned enzyme.
[0051]
In addition, the above-mentioned disrupted product of
microorganism, crude enzyme or purified enzyme may be
immobilized on a water-insoluble carrier, gel and the like,
and used as the above-mentioned enzyme.
CDP-choline can be produced by bringing the above-
mentioned enzyme in contact with the precursor of UTP and
choline or phosphorylcholine or a salt thereof in the medium
to produce and accumulate CDP-choline, and recovering CDP-
choline from the medium.
[0052]
A specific method of producing and accumulating CDP-
21

choline comprises mixing the above-mentioned enzyme and the
precursor of UTP and choline or phosphorylcholine or a salt
thereof in a medium, adding other components as necessary to
the obtained mixture and maintaining the mixture at 20 - 50°C
for 2-48 hours while keeping pH 5 - 11, more preferably 6 -
10.
The amount of the CDP-producing enzyme to be used varies
depending on the specific activity and the like of the enzyme.
For example, when the crude enzyme is used as the enzyme, it
is preferably used in an amount of 1 \iq - 500 mg, preferably 10
jig - 300 mg, relative to 1 mg of choline chloride.
[0053]
The precursor of UTP, choline, phosphorylcholine, a salt
thereof to be added and other components to be added as
necessary when producing and accumulating CDP-choline using
the CDP-choline-producing enzyme are similar to those used for
producing and accumulating CDP-choline using the culture and
the like of the microorganism as mentioned above. Furthermore,
adenosine-5'-triphosphate and the like may be added as
necessary as an energy donor, and 5-phosphoribosyl diphosphate
may be further added.
[0054]
As the medium in which the CDP-choline-producing enzyme
is brought into contact with the precursor of UTP and choline
or phosphorylcholine or a salt thereof, the medium used for
culturing the microorganism to be used as the biocatalyst, the
culture, the supernatant of the culture and the like of the
microorganism may be used, or an aqueous medium may also be
used.
Examples of the aqueous medium include water, buffers
such as phosphate buffer, HEPES (N-2-hydroxyethylpiperazine-N-
ethanesulfonic acid) buffer, tris
[tris(hydroxymethyl)aminomethane] hydrochloride and the like.
[0055]
For preparing the CDP-choline solution from the medium in
22

which CDP-choline has been produced and accumulated as
mentioned above, a solid may be separated and removed from the
medium according to a method using membrane separation,
filtration, centrifugation and the like.
Examples of the nucleic acid analogue contained in the
CDP-choline solution prepared by the above-mentioned method
include uracil, UTP and the like.
CDP-choline and nucleic acid analogs can be analyzed by a
conventional method using high performance liquid
chromatography (UV detection).
[0056]
The present invention is explained in more detail in
the following by referring to Examples, which are not to be
construed as limitative.
Example 1
Purification of CDP-choline using strongly acidic cation
exchange resin
CDP-choline (50 g; manufactured by Wako Pure Chemical
Industries, Ltd.), uracil (2 g; manufactured by Nacalai
Tesque) and UTP (1 g; manufactured by Nacalai Tesque) were
dissolved in water to prepare 5 L of CDP-choline solution. The
solution was adjusted to pH 3.0 with sulfuric acid and applied
to a column packed with a strongly acidic cation exchange
resin Diaion PK208 (H-type) (10 L) with a crosslinking degree
of 4%. Subsequently, water was applied, and a fraction wherein
each concentration of uracil and UTP was less than 0.1% (w/w)
relative to CDP-choline was obtained as a result of analysis
by high performance liquid chromatography. The CDP-choline
fraction was concentrated to 100 mL, and ethanol (350 mL) was
gradually added. The precipitated crystals were collected by
filtration, washed with 100% ethanol solution, and dried under
reduced pressure at 20°C for 3 days. As the result, 40 g of
CDP-choline crystals, wherein each concentration of uracil and
UTP was less than 0.1% (w/w) relative to CDP-choline, were
obtained.
23

Example 2
Purification of CDP-choline from culture of microorganism
having an activity to produce CDP-choline
Escherichia coli MM294/pCKG55 strain (FERM BP-3717)
having enzyme activities of PyrG, CCT and CKI was inoculated
to a test tube containing L medium (10 mL) [liquid medium
containing bactotripton (10 g/L; manufactured by Difco), yeast
extract (5 g/L; manufactured by Difco) and NaCl (5 g/L) and
adjusted to pH 7.2] supplemented with ampicillin (50 jig/mL) ,
and cultured with shaking at 25°C, 300 rpm for 24 hours. The
culture thus obtained (20 mL) was inoculated to 2 L of conical
flask with baffles containing L medium (400 mL) supplemented
with ampicillin (50 |j.g/mL) and cultured with rotary shaking at
25°C, 190 rpm for 16 hours. The culture thus obtained (125 mL)
was inoculated to a 5 L culture bath containing a liquid
medium (2.5 L; nonadjusted pH) with a composition of glucose
(5 g/L; separately sterilized), peptone (5 g/L; manufactured
by Kyokuto Pharmaceutical Industrial), Na2HP04 (6 g/L), KH2P04
(3 g/L), NaCl (5 g/L), NH4C1 (1 g/L), MgS04-7H20 (250 mg/L;
separately sterilized) and vitamin Bl (4 [J.g/L; separately
sterilized), and cultured with shaking at 600 rpm under
culture condition of airflow 2.5 L/minute, at 25°C for 11 hours
and then at 32°C for 13 hours, while adjusting the mixture to
pH 7.0 with 14% aqueous ammonia. During culturing, a feed
solution having a composition of glucose (167 g/L), peptone
(167 g/L) was fed with a peristaltic pump at a rate of 30 mL/
hours during the period of from 11 hours to 24 hours from the
start of the culturing.
[0059]
On the other hand, Corynebacterium*ammoniagenes ATCC21170
strain having an activity to produce UTP from orotic acid was
inoculated to a test tube containing a liquid medium (10 mL)
having a composition of glucose (50 g/L), polypeptone (10 g/L;
manufactured by Daigo Eiyo Chemicals), yeast extract (10 g/L;
manufactured by Daigo Eiyo Chemicals), urea (5 g/L), (NH4)2S04
24

(5 g/L), KH2P04 (1 g/L) , K2HP04 (3 g/L) , MgS04'7H20 (1 g/L) ,
CaCl2-2H20 (0.1 g/L), FeS04-7H20 (10 mg/L), ZnS04-7H20 (10 mg/L) ,
MnS04-4 - 6H20 (20 mg/L), L-cysteine (20 mg/L), calcium D-
pantothenate (10 mg/L), vitamin Bl (5 mg/L), nicotinic acid (5
mg/L) and biotin (30 jig/L; adjusted to pH 7.2 with sodium
hydroxide), and cultured with reciprocal shaking at 300 rpm at
28°C for 24 hours. The culture thus obtained (20 mL) was
inoculated to a 2 L conical flask with baffle containing a
liquid medium (230 mL) having the same composition as the
above-mentioned, and cultured with rotary shaking at 190 rpm
at 28°C for 24 hours. The culture thus obtained (250 mL) was
inoculated to a 5 L culture bath containing a liquid medium
(2.5 L) having a composition of glucose (100 g/L), meat
extract (10 g/L), polypeptone (10 g/L), KH2P04 (1 g/L), K2HP04
(1 g/L), MgS04.7H20 (1 g/L), CaCl2-2H20 (0.1 g/L), FeS04-7H20 (20
mg/L), ZnS04-7H20 (10 mg/L), MnS04-4 - 6H20 (20 mg/L), p-alanine
(15 mg/L), L-cysteine (20 mg/L), biotin (100 p,g/L) , urea (2
g/L; separately sterilized) and vitamin Bl (5 mg/L; separately
sterilized) (adjusted to pH 7.2 with sodium hydroxide) and
seed-cultured with shaking at 600 rpm at 32°C under culture
condition of airflow 2.5 L/minute while adjusting the mixture
to pH 6.8 with concentrated aqueous ammonia. At the time point
when glucose in the supernatant of the above-mentioned seed-
culture medium was consumed, the culture (350 mL) was
aseptically collected, inoculated to a 5 L culture bath
containing a liquid medium (2.5 L) having a composition of
glucose (180 g/L), KH2P04 (10 g/L), K2HP04 (10 g/L), MgS04-7H20
(10 g/L), CaCl2-2H20 (0.1 g/L), FeS04-7H20 (20 mg/L), ZnS04-7H20
(10 mg/L), MnS04-4 - 6H20 (20 mg/L; separately sterilized), 0-
alanine (15 mg/L), L-cysteine (20 mg/L), sodium glutamate (1
g/L) , biotin (100 \iq/L) , urea (2 g/L; separately sterilized)
and vitamin Bl (5 mg/L; separately sterilized) (adjusted to pH
7.2 with sodium hydroxide) and main-cultured with shaking at
600 rpm at 32°C under culture condition of airflow 2.5 L/minute
while adjusting the mixture to pH 6.8 with concentrated
25

aqueous ammonia. The culturing was ceased at the time point
when glucose in the culture supernatant was consumed.
[0060]
The culture (360 mL) of Escherichia coli MM294/pCKG55
strain and the culture (360 mL) of Corynebacterium-ammoniagenes
ATCC21170 strain thus obtained were placed in a 2 L culture
bath, glucose (100 g/L), orotic acid (10 g/L), choline
chloride (8.4 g/L), MgS04-7H20 (5 g/L) and xylene (20 mL/L)
were added thereto, and distilled water was added to allow the
total amount to be 800 mL. The mixture was reacted with
shaking at 800 rpm at 32°C under the condition of airflow 0.8
L/minute while adjusting the mixture to pH 7.2 with ION sodium
hydroxide. During the reaction, KH2P04 was added as
appropriate to keep the concentration of phosphoric acid in
the supernatant of the reaction mixture at 1 - 5 g/L as KH2P04.
The reaction was carried out for 23 hours to obtain 11.0 g/L
of CDP-choline.
[0061]
Four batches of the above-mentioned reaction mixture were
adjusted to pH 1.0 with sulfuric acid, cells were separated by
centrifugation (7000 rpm, 10 min), and water was added to the
obtained supernatant to make the total amount 6 L (CDP-choline
6.0 g/L, uracil 0.5 g/L, UTP 1.0 g/L). This
cytidinediphosphoric choline solution was applied to a column
packed with a strongly acidic cation exchange resin Diaion
SK104 (H-type, 10 L) having a crosslinking degree of 4%.
Subsequently, water was applied, and a fraction, in which each
concentration of uracil, UTP was less than 0.1%(w/w) relative
to CDP-choline (analyzed by high performance liquid
chromatography) was collected. The CDP-choline fraction was
decolorized using activated carbon, and then concentrated to
100 mL. Ethanol (350 mL) was gradually added to the
concentrated solution, and the precipitated crystals were
collected by filtration. The obtained crystals were washed
with 100% ethanol solution, and then dried under reduced
26

pressure at 20°C for 3 days. As a result, CDP-choline crystals
(18 g), in which each concentration of uracil, UTP was less
than 0.1% (w/w) relative to CDP-choline, were obtained.
Example 3
Purification of CDP-choline sodium salt from a culture of
microorganism having an ability to produce CDP-choline
Four batches of the reaction mixture obtained in the same
manner as in Example 2 were adjusted to pH 1.0 with sulfuric
acid, cells were separated by centrifugation (7000 rpm, 10
min), and water was added to the obtained supernatant to make
the total amount 6 L (CDP-choline 6.0 g/L, uracil 0.5 g/L, UTP
1.0 g/L). This cytidine diphosphate choline solution was
applied to a column packed with a strongly acidic cation
exchange resin Diaion SK104 (H-type, 10 L) having a
crosslinking degree of 4%. Subsequently, water was applied,
and a fraction, in which each concentration of uracil, UTP was
less than 0.1%(w/w) relative to CDP-choline (analyzed by high
performance liquid chromatography) was collected. The CDP-
choline fraction was adjusted to pH 7.5 with sodium hydroxide,
decolorized with activated carbon, and then concentrated to
100 mL. Ethanol (400 mL) was gradually added to the
concentrated solution, and the precipitated crystals were
collected by filtration. The obtained crystals were washed
with 100% ethanol solution, and then dried under reduced
pressure at 20°C for 3 days. As a result, CDP-choline sodium
salt crystals (20 g), in which each concentration of uracil,
UTP was less than 0.1% (w/w) relative to CDP-choline, were
obtained.
[0063]
From the foregoing results, it has been clarified that
CDP-choline or a salt thereof free from impurity can be
obtained conveniently by treating a CDP-choline solution
containing a nucleic acid analogue only once with a strongly
acidic cation exchange resin.
Industrial Applicability
27

[0064]
According to the present invention, CDP-choline and a
salt thereof are provided at a low cost.
28

Claims
1. A method of purifying cytidine diphosphate choline
(hereinafter abbreviated as CDP-choline), which comprises
contacting a CDP-choline solution containing a nucleic acid
analogue and having a pH of not less than 0.5 and not more
than 5.0 with an H-type strongly acidic cation exchange resin,
and eluting CDP-choline adsorbed onto the resin with water or
an aqueous solution having an ion concentration of not more
than 0.1 mol/L to separate and purify CDP-choline.
2. The method of claim 1, wherein the CDP-choline solution is
prepared from a resulting medium containing CDP-choline, which
has been produced and accumulated therein by placing a
biocatalyst having an activity to produce CDP-choline from a
precursor of uridine-5'-triphosphate (hereinafter abbreviated
as UTP) and choline or phosphorylcholine in an aqueous medium
together with the precursor of UTP and choline or
phosphorylcholine or a salt thereof.
3. The method of claim 2, wherein the biocatalyst comprises a
culture or a treated culture of a microorganism capable of
producing UTP from a precursor of UTP, and a culture or a
treated culture of a microorganism capable of producing CDP-
choline from UTP and choline or phosphorylcholine.

4. The method of claim 2, wherein the biocatalyst comprises an
enzyme that catalyzes a reaction to produce CDP-choline from a
precursor of UTP and choline or phosphorylcholine.
5. The method of claim 4, wherein the enzyme that catalyzes a
reaction to produce CDP-choline is an enzyme selected from the
group consisting of orotate phosphoribosyl transferase,
orotidine-5'-monophosphate decarboxylase, uridine
phosphorylase, uracil phosphoribosyltransferase, uridine
29

kinase, uridylate and cytidylate kinases, nucleoside
diphosphate kinase, cytidine-5'-triphosphate synthase, choline
phosphate cytidyltransferase and choline kinase.
30
6. The method of any of claims 1 to 5, wherein the nucleic
acid analogue is selected from uracil and UTP.

A method of purifying cytidine diphosphate choline, which
comprises contacting a cytidine diphosphate choline solution
containing a nucleic acid analogue and having a pH of not less
than 0.5 and not more than 5.0 with an H-type strongly acidic
cation exchange resin, and eluting cytidine diphosphate
choline adsorbed onto the resin with water or an aqueous
solution having an ion concentration of not more than 0.1
mol/L to separate and purify the cytidine diphosphate choline.

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=lyHVubLtmYAWfOQUvnlBGQ==&loc=wDBSZCsAt7zoiVrqcFJsRw==

« Previous Patent

Next Patent »

Patent Number

270397

Indian Patent Application Number

634/KOLNP/2008

PG Journal Number

52/2015

Publication Date

25-Dec-2015

Grant Date

17-Dec-2015

Date of Filing

13-Feb-2008

Name of Patentee

KYOWA HAKKO BIO CO., LTD

Applicant Address

1-6-1, OHTEMACHI, CHIYODA-KU, TOKYO

Inventors:

#	Inventor's Name	Inventor's Address
1	HIDEKI MURATA	C/O. HOFU PLANT KYOWA HAKKO KOGYO CO., LTD. 1-1, KYOWA-CHO, HOFU-SHI, YAMAGUCHI 747-8522
2	MICHIO SHIOMI	9673, KIDO, YOKOSHIBAHIKARI-MACHI, YAMABU-GUN, CHIBA 289-1726
3	TSUYOSHI MOKUDAI	C/O. HOFU PLANT KYOWA HAKKO KOGYO CO., LTD. 1-1, KYOWA-CHO, HOFU-SHI, YAMAGUCHI 747-8522

PCT International Classification Number

C07H 19/10

PCT International Application Number

PCT/JP2006/315802

PCT International Filing date

2006-08-10

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	2005-231958	2005-08-10	Japan