WO1999049073A1 - Procede de production de cytidine 5'-diphosphate choline - Google Patents
Procede de production de cytidine 5'-diphosphate choline Download PDFInfo
- Publication number
- WO1999049073A1 WO1999049073A1 PCT/JP1999/001312 JP9901312W WO9949073A1 WO 1999049073 A1 WO1999049073 A1 WO 1999049073A1 JP 9901312 W JP9901312 W JP 9901312W WO 9949073 A1 WO9949073 A1 WO 9949073A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- choline
- reaction
- cdp
- cytidine
- cct
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 27
- RZZPDXZPRHQOCG-OJAKKHQRSA-O CDP-choline(1+) Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OCC[N+](C)(C)C)O[C@H]1N1C(=O)N=C(N)C=C1 RZZPDXZPRHQOCG-OJAKKHQRSA-O 0.000 title abstract 3
- 238000006243 chemical reaction Methods 0.000 claims abstract description 40
- 229960001231 choline Drugs 0.000 claims abstract description 14
- 108010018888 Choline kinase Proteins 0.000 claims abstract description 13
- 229950004354 phosphorylcholine Drugs 0.000 claims abstract description 13
- 210000005253 yeast cell Anatomy 0.000 claims abstract description 13
- 102000002745 Choline Kinase Human genes 0.000 claims abstract description 12
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 claims abstract description 10
- YHHSONZFOIEMCP-UHFFFAOYSA-O phosphocholine Chemical compound C[N+](C)(C)CCOP(O)(O)=O YHHSONZFOIEMCP-UHFFFAOYSA-O 0.000 claims abstract description 7
- 102000003960 Ligases Human genes 0.000 claims abstract description 3
- 108090000364 Ligases Proteins 0.000 claims abstract description 3
- 108090000790 Enzymes Proteins 0.000 claims description 25
- 102000004190 Enzymes Human genes 0.000 claims description 24
- 238000004519 manufacturing process Methods 0.000 claims description 11
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 claims description 7
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 claims description 7
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 claims description 7
- 150000004712 monophosphates Chemical class 0.000 claims description 2
- 125000002740 cytidyl group Chemical group 0.000 claims 1
- 239000001177 diphosphate Substances 0.000 claims 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims 1
- 235000011180 diphosphates Nutrition 0.000 claims 1
- 239000001226 triphosphate Substances 0.000 claims 1
- 235000011178 triphosphate Nutrition 0.000 claims 1
- 230000006378 damage Effects 0.000 abstract description 3
- 208000022540 Consciousness disease Diseases 0.000 abstract description 2
- DJJCXFVJDGTHFX-UHFFFAOYSA-N Uridinemonophosphate Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-UHFFFAOYSA-N 0.000 abstract description 2
- 210000004556 brain Anatomy 0.000 abstract description 2
- PCDQPRRSZKQHHS-UHFFFAOYSA-N Cytidine 5'-triphosphate Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 PCDQPRRSZKQHHS-UHFFFAOYSA-N 0.000 abstract 1
- 208000006011 Stroke Diseases 0.000 abstract 1
- 208000027418 Wounds and injury Diseases 0.000 abstract 1
- 208000014674 injury Diseases 0.000 abstract 1
- 230000001105 regulatory effect Effects 0.000 abstract 1
- DJJCXFVJDGTHFX-ZAKLUEHWSA-N uridine-5'-monophosphate Chemical compound O[C@@H]1[C@@H](O)[C@H](COP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-ZAKLUEHWSA-N 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 30
- RZZPDXZPRHQOCG-OJAKKHQRSA-M CDP-choline(1-) Chemical compound O[C@@H]1[C@H](O)[C@@H](COP([O-])(=O)OP([O-])(=O)OCC[N+](C)(C)C)O[C@H]1N1C(=O)N=C(N)C=C1 RZZPDXZPRHQOCG-OJAKKHQRSA-M 0.000 description 25
- 239000000243 solution Substances 0.000 description 24
- 229940088598 enzyme Drugs 0.000 description 23
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 20
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 18
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 18
- 210000004027 cell Anatomy 0.000 description 16
- 241000588724 Escherichia coli Species 0.000 description 15
- 238000002360 preparation method Methods 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 13
- 230000000694 effects Effects 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 10
- 239000008103 glucose Substances 0.000 description 10
- 238000003786 synthesis reaction Methods 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 229910001629 magnesium chloride Inorganic materials 0.000 description 9
- YHHSONZFOIEMCP-UHFFFAOYSA-N 2-(trimethylazaniumyl)ethyl hydrogen phosphate Chemical compound C[N+](C)(C)CCOP(O)([O-])=O YHHSONZFOIEMCP-UHFFFAOYSA-N 0.000 description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 108091008146 restriction endonucleases Proteins 0.000 description 8
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 244000005700 microbiome Species 0.000 description 7
- 102000012410 DNA Ligases Human genes 0.000 description 6
- 108010061982 DNA Ligases Proteins 0.000 description 6
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 6
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 6
- 229960000723 ampicillin Drugs 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 230000000813 microbial effect Effects 0.000 description 6
- DJJCXFVJDGTHFX-XVFCMESISA-N uridine 5'-monophosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-XVFCMESISA-N 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 238000007796 conventional method Methods 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 4
- 238000010369 molecular cloning Methods 0.000 description 4
- 229910000160 potassium phosphate Inorganic materials 0.000 description 4
- 235000011009 potassium phosphates Nutrition 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 239000011541 reaction mixture Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 3
- 230000004544 DNA amplification Effects 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- 108010010803 Gelatin Proteins 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 239000013611 chromosomal DNA Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 101150097077 cki gene Proteins 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 239000008273 gelatin Substances 0.000 description 3
- 229920000159 gelatin Polymers 0.000 description 3
- 235000019322 gelatine Nutrition 0.000 description 3
- 235000011852 gelatine desserts Nutrition 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 description 2
- QAIRZQBZKJPMHC-UHFFFAOYSA-J 2-hydroxyethyl(trimethyl)azanium phosphonato phosphate Chemical compound C[N+](C)(C)CCO.C[N+](C)(C)CCO.C[N+](C)(C)CCO.C[N+](C)(C)CCO.[O-]P([O-])(=O)OP([O-])([O-])=O QAIRZQBZKJPMHC-UHFFFAOYSA-J 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 235000019743 Choline chloride Nutrition 0.000 description 2
- ZWIADYZPOWUWEW-UHFFFAOYSA-N Cytidine 5'-diphosphate Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(COP(O)(=O)OP(O)(O)=O)O1 ZWIADYZPOWUWEW-UHFFFAOYSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 241000235070 Saccharomyces Species 0.000 description 2
- HSCJRCZFDFQWRP-JZMIEXBBSA-N UDP-alpha-D-glucose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-JZMIEXBBSA-N 0.000 description 2
- HSCJRCZFDFQWRP-UHFFFAOYSA-N Uridindiphosphoglukose Natural products OC1C(O)C(O)C(CO)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-UHFFFAOYSA-N 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 229960003178 choline chloride Drugs 0.000 description 2
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 description 2
- ZWIADYZPOWUWEW-ZAKLUEHWSA-N cytidine-5'-diphosphate Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO[P@](O)(=O)OP(O)(O)=O)O1 ZWIADYZPOWUWEW-ZAKLUEHWSA-N 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000003505 heat denaturation Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000012134 supernatant fraction Substances 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 208000035404 Autolysis Diseases 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 206010057248 Cell death Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 102100031065 Choline kinase alpha Human genes 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 241000235035 Debaryomyces Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010019196 Head injury Diseases 0.000 description 1
- 101000777314 Homo sapiens Choline kinase alpha Proteins 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 241000235017 Zygosaccharomyces Species 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 150000002232 fructoses Chemical class 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- -1 isopropyl- Chemical group 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 101150054232 pyrG gene Proteins 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 230000028043 self proteolysis Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/305—Pyrimidine nucleotides
Definitions
- the present invention head trauma, consciousness disorders associated with brain surgery, cytidine 5 1 are found using the improved treatment such as stroke - Ru der method of manufacturing a diphosphate choline (CDP-choline).
- CDP-choline diphosphate choline
- CCT choline kinase
- CKI choline kinase
- cytidine 5 Ichito only Urijin 5 1 coexist Li phosphate synthetase (PyrG) - monophosphate (5 1 - UMP) and choline
- the present invention provides yeast cells, 5 1 - a UMP and choline, CCT, Ru der relates CDP- choline manufacturing method characterized by reacting in the presence of CKI and PyrG.
- the present invention is a yeast cell, 5 1 - a UMP and phosphorylcholine
- Ru der relates CDP- choline manufacturing method characterized by reacting in the presence of CCT and PyrG.
- Figure 1 shows the effect of reaction temperature on CDP-choline productivity and the composition of the reaction mixture.
- each substrate is preferably in the range of about 1 to 200 mM, particularly about 10 to 100 mM.
- DNA preparation, restriction enzyme digestion, DNA ligation using T4 DNA ligase, and E. coli transformation were all performed by “Molecular cloning”.
- Amplification of CCT gene by PCR is as follows: Reaction solution 10 ( ⁇ 1 [50 mM chloride, 10 mM Tris-HCl (pH 8.3), 1.5 mM magnesium chloride, 0.001% gelatin, 0.5 g of temperate DNA, primer DNA (A) and (B) in each 0.2 lambda M, AmpliTaq DNA polymerase La one peptidase 2.5 Yuni' preparative], using a Perkin-Elmer Cetus Instrument Co., Ltd.
- the reaction solution was treated with a mixture of phenol nocroform-form (1: 1), and a two-fold solution of ethanol was added to the water-soluble fraction to precipitate DNA.
- the DNA collected by precipitation was separated by agarose gel electrophoresis according to the method described in the literature (Molecular cloning, described above), and a DNA fragment corresponding to 2.0 kb was purified.
- the DNA was cut with restriction enzymes Xbal and Pstl, and plasmid pTrc99A was digested with restriction enzymes Xbal and Pstl.
- pTrc-CKI is obtained by inserting an Xbal-Pstl DNA fragment containing the yeast CKI gene into an Xbal-Pstl cleavage site downstream of the trc promoter of pTrc99A.
- Plasmid pTrc-CCT was digested with restriction enzymes Ncol and O ⁇ Xbal, and the Ncol-Xbal fragment containing the CCT gene was separated and purified. The fragment was ligated with plasmid pTrc-CKI DNA cut with Ncol and al using T4 DNA ligase, and Escherichia coli JM109 was transformed using the ligation reaction solution. Plasmid pTrc-CCK was isolated from the obtained ampicillin-resistant transformant. pTrc-CCK is obtained by ligating and inserting yeast CCT and CKI genes downstream of the trc promoter of pTrc99A.
- Escherichia coli JM109 harboring plasmid pTrc-CCK was inoculated into 300 ml of 2 XYT medium containing 100 g / ml ampicillin and cultured with shaking at 37 ° C. 4
- IPTG isopropyl-/?-D-thiogalactovyranoside
- the unit (unit) of each enzyme was measured and calculated by the following method.
- the amount of CDP-choline in the reaction solution is analyzed by (HPLC).
- the activity to produce 1 mole of CDP-choline per minute at 37 ° C is defined as 1 unit (unit).
- the measurement of PyrG activity and the calculation of units were performed by the following methods. That is, an enzyme preparation was added to a 200 mM Tris-HCl buffer (pH 7.8) containing 5 mM UTP, 5 mM phosphocholine, 5 mM ATP, 10 mM magnesium chloride, 50 mM ammonium sulfate, and 1 unit / ml CCT. Perform the reaction at 37 ° C, and stop the reaction by heat treatment at 100 ° C for 1 minute. The amount of CDP-choline in the reaction solution is determined by the HPLC method. The activity to produce 1 ⁇ mol of CDP-choline per minute at 37 ° C is defined as 1 unit (unit).
- the present invention provides a simple and efficient method for coexisting CCT, CKI (not required when phosphorylcholine is used) and PyrG in a reaction system of yeast cells, 5 "-UMP, and choline or phosphorylcholine.
- the reaction temperature within the range of about 20-30 ° C, the synthesis yield of CDP-choline can be significantly increased, while the production of unintended products such as UDP-glucose can be kept low.
- by dividing and adding the energy source it is possible to further improve the productivity of CDP-choline.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Procédé de production efficace de cytidine 5'-diphosphate choline à utiliser pour l'amélioration de troubles de la conscience associés, entre autres, aux coups de chaleur ou à une opération du cerveau, à une attaque cérébrale, etc. Ledit procédé consiste à ajouter de la choline-phosphate citidyltransférase, de la choline kinase (inutile lorsque de la phosphorylcholine est utilisée) et de la cytidine 5'-triphosphate synthétase à un système réactionnel contenant des cellules de levure, de l'uridine 5'-monophosphate et de la choline ou de la phosphorylcholine. En régulant la température de réaction, de sorte qu'elle soit de 20 à 30 °C environ, et en ajoutant une source d'énergie dans certaines parties, en particulier, on peut encore accroître la productivité de la cytidine 5'-diphosphate choline.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10/92626 | 1998-03-20 | ||
| JP9262698 | 1998-03-20 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1999049073A1 true WO1999049073A1 (fr) | 1999-09-30 |
Family
ID=14059664
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/JP1999/001312 WO1999049073A1 (fr) | 1998-03-20 | 1999-03-17 | Procede de production de cytidine 5'-diphosphate choline |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO1999049073A1 (fr) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003095660A1 (fr) | 2002-05-08 | 2003-11-20 | Kyowa Hakko Kogyo Co., Ltd. | Procede de production de cytidine 5'-diphosphate choline |
| WO2007018259A1 (fr) | 2005-08-10 | 2007-02-15 | Kyowa Hakko Kogyo Co., Ltd. | Procédé de purification de la cytidine diphosphocholine |
| CN111808899A (zh) * | 2020-08-31 | 2020-10-23 | 宁波酶赛生物工程有限公司 | 一种胞磷胆碱钠的合成方法 |
| CN116790466A (zh) * | 2023-07-19 | 2023-09-22 | 山东理工大学 | 一种工程改造枯草芽孢杆菌发酵生产胞磷胆碱的方法 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5127754B1 (fr) * | 1969-12-19 | 1976-08-14 | ||
| JPH05276974A (ja) * | 1992-01-30 | 1993-10-26 | Kyowa Hakko Kogyo Co Ltd | シチジンジリン酸コリンの製造法 |
-
1999
- 1999-03-17 WO PCT/JP1999/001312 patent/WO1999049073A1/fr active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5127754B1 (fr) * | 1969-12-19 | 1976-08-14 | ||
| JPH05276974A (ja) * | 1992-01-30 | 1993-10-26 | Kyowa Hakko Kogyo Co Ltd | シチジンジリン酸コリンの製造法 |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003095660A1 (fr) | 2002-05-08 | 2003-11-20 | Kyowa Hakko Kogyo Co., Ltd. | Procede de production de cytidine 5'-diphosphate choline |
| WO2007018259A1 (fr) | 2005-08-10 | 2007-02-15 | Kyowa Hakko Kogyo Co., Ltd. | Procédé de purification de la cytidine diphosphocholine |
| US8303820B2 (en) | 2005-08-10 | 2012-11-06 | Kyowa Hakko Bio Co., Ltd. | Method of purifying cytidine diphosphate choline |
| CN111808899A (zh) * | 2020-08-31 | 2020-10-23 | 宁波酶赛生物工程有限公司 | 一种胞磷胆碱钠的合成方法 |
| CN116790466A (zh) * | 2023-07-19 | 2023-09-22 | 山东理工大学 | 一种工程改造枯草芽孢杆菌发酵生产胞磷胆碱的方法 |
| CN116790466B (zh) * | 2023-07-19 | 2023-11-03 | 山东理工大学 | 一种工程改造枯草芽孢杆菌发酵生产胞磷胆碱的方法 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4505011B2 (ja) | ３’−ホスホアデノシン−５’−ホスホ硫酸の酵素合成法 | |
| KR970010134B1 (ko) | 뉴클레오시드의 제조법 | |
| US6040158A (en) | Process for preparing sugar nucleotide | |
| JP3764755B2 (ja) | 「アデノシン５’−三リン酸の製造法及びその応用」 | |
| JP3822006B2 (ja) | シチジン５’−ジリン酸コリンの製造法 | |
| WO1999049073A1 (fr) | Procede de production de cytidine 5'-diphosphate choline | |
| JPWO2004009830A1 (ja) | Ｃｍｐ−ｎ−アセチルノイラミン酸の製造法 | |
| JP3833584B2 (ja) | Ｃｍｐ−ｎ−アセチルノイラミン酸の製造法 | |
| JP4173815B2 (ja) | ２’−デオキシグアノシンの製造法 | |
| JP3545425B2 (ja) | ウリジン二リン酸―ｎ―アセチルグルコサミンの製造法 | |
| EP1233072B1 (fr) | Nouvelle utilisation de l'uridine-diphosphate-glucose 4-epimerase | |
| JPWO2005105991A1 (ja) | 放線菌由来のａｍｐデアミナーゼ及びその利用 | |
| JP3729712B2 (ja) | デオキシヌクレオシドの酵素的製造法 | |
| JP2003093091A5 (fr) | ||
| JP5068956B2 (ja) | デオキシリボヌクレオシド一リン酸からのデオキシリボヌクレオシド三リン酸の製造方法 | |
| WO1985004187A1 (fr) | Procede de preparation d'acide 5'-guanylique | |
| KR100680765B1 (ko) | 3'-아미노-2',3'-디데옥시구아노신의 제조 방법 | |
| JP4010996B2 (ja) | デオキシヌクレオシド５’−モノリン酸の製造法 | |
| JP2002085087A (ja) | シチジン５’−トリリン酸の製造法及びその応用 | |
| JP2001169797A (ja) | Ｓ―アデノシル−ｌ−メチオニンの酵素的製造法 | |
| JP2000041695A (ja) | Ｓ―アデノシル−ｌ−メチオニンの酵素的製造法 | |
| JP4509447B2 (ja) | 高純度グアノシン５′−ジリン酸フコースおよびその製造法 | |
| JP2000217593A (ja) | シチジン５’−トリリン酸の酵素的製造法およびその応用 | |
| JPH05219978A (ja) | 核酸関連物質の酵素的製造法及びそれに使用する酵素調製物 | |
| CN117143942A (zh) | 3’-氨基-2’,3’-双脱氧鸟苷5’-三磷酸的合成方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| AK | Designated states |
Kind code of ref document: A1 Designated state(s): CA CN JP KR US |
|
| AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
| DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
| NENP | Non-entry into the national phase |
Ref country code: KR |
|
| 122 | Ep: pct application non-entry in european phase |