JPH0337919B2 - - Google Patents
Info
- Publication number
- JPH0337919B2 JPH0337919B2 JP57100832A JP10083282A JPH0337919B2 JP H0337919 B2 JPH0337919 B2 JP H0337919B2 JP 57100832 A JP57100832 A JP 57100832A JP 10083282 A JP10083282 A JP 10083282A JP H0337919 B2 JPH0337919 B2 JP H0337919B2
- Authority
- JP
- Japan
- Prior art keywords
- reaction
- ribavirin
- microorganisms
- ribose
- triazole
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 244000005700 microbiome Species 0.000 claims description 50
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 claims description 37
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 claims description 35
- 229960000329 ribavirin Drugs 0.000 claims description 35
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 26
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 claims description 20
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims description 20
- 238000004519 manufacturing process Methods 0.000 claims description 15
- ZEWJFUNFEABPGL-UHFFFAOYSA-N 1,2,4-triazole-3-carboxamide Chemical compound NC(=O)C=1N=CNN=1 ZEWJFUNFEABPGL-UHFFFAOYSA-N 0.000 claims description 13
- 150000003839 salts Chemical class 0.000 claims description 10
- 239000012736 aqueous medium Substances 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 241000589565 Flavobacterium Species 0.000 claims description 4
- 230000009471 action Effects 0.000 claims description 4
- SJCDBQHCQSIZHN-UHFFFAOYSA-N 1,2-dihydrotriazole-3-carboxamide Chemical compound NC(=O)N1NNC=C1 SJCDBQHCQSIZHN-UHFFFAOYSA-N 0.000 claims description 3
- 241000186321 Cellulomonas Species 0.000 claims description 3
- 241000588914 Enterobacter Species 0.000 claims description 3
- 241000588698 Erwinia Species 0.000 claims description 3
- 241000588722 Escherichia Species 0.000 claims description 3
- 241000588748 Klebsiella Species 0.000 claims description 3
- 241000589516 Pseudomonas Species 0.000 claims description 3
- 241000607598 Vibrio Species 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 description 70
- 238000000034 method Methods 0.000 description 47
- 230000001580 bacterial effect Effects 0.000 description 41
- 210000004027 cell Anatomy 0.000 description 39
- 239000000758 substrate Substances 0.000 description 38
- 239000000243 solution Substances 0.000 description 27
- 102000004190 Enzymes Human genes 0.000 description 23
- 108090000790 Enzymes Proteins 0.000 description 23
- 229940088598 enzyme Drugs 0.000 description 23
- 238000011282 treatment Methods 0.000 description 22
- 239000000126 substance Substances 0.000 description 15
- -1 triazole compound Chemical class 0.000 description 15
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 14
- 238000006911 enzymatic reaction Methods 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 239000006285 cell suspension Substances 0.000 description 10
- 238000005119 centrifugation Methods 0.000 description 10
- 238000012258 culturing Methods 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 8
- 230000000813 microbial effect Effects 0.000 description 8
- 238000000926 separation method Methods 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 7
- 239000007864 aqueous solution Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 6
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 5
- 238000012136 culture method Methods 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000003960 organic solvent Substances 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 5
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 4
- 229930010555 Inosine Natural products 0.000 description 4
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 4
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 150000002148 esters Chemical class 0.000 description 4
- 229960003786 inosine Drugs 0.000 description 4
- 229910052700 potassium Inorganic materials 0.000 description 4
- 239000011591 potassium Substances 0.000 description 4
- 230000035484 reaction time Effects 0.000 description 4
- 150000003290 ribose derivatives Chemical class 0.000 description 4
- 229910052708 sodium Inorganic materials 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 235000000346 sugar Nutrition 0.000 description 4
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 3
- OQRXBXNATIHDQO-UHFFFAOYSA-N 6-chloropyridine-3,4-diamine Chemical compound NC1=CN=C(Cl)C=C1N OQRXBXNATIHDQO-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 108010009736 Protein Hydrolysates Proteins 0.000 description 3
- 102100036286 Purine nucleoside phosphorylase Human genes 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229910021529 ammonia Inorganic materials 0.000 description 3
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 3
- 239000002585 base Substances 0.000 description 3
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 3
- 229910052791 calcium Inorganic materials 0.000 description 3
- 239000011575 calcium 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
- 238000007796 conventional method Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N hydrochloric acid Substances Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 229910052749 magnesium Inorganic materials 0.000 description 3
- 239000011777 magnesium Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 description 3
- 150000002823 nitrates Chemical class 0.000 description 3
- 108010009099 nucleoside phosphorylase Proteins 0.000 description 3
- 229940085991 phosphate ion Drugs 0.000 description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000012264 purified product Substances 0.000 description 3
- 239000002342 ribonucleoside Substances 0.000 description 3
- 150000008163 sugars Chemical class 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 2
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- XTHFKEDIFFGKHM-UHFFFAOYSA-N Dimethoxyethane Chemical compound COCCOC XTHFKEDIFFGKHM-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 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 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- AMQJEAYHLZJPGS-UHFFFAOYSA-N N-Pentanol Chemical compound CCCCCO AMQJEAYHLZJPGS-UHFFFAOYSA-N 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 241000191940 Staphylococcus Species 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 229950006790 adenosine phosphate Drugs 0.000 description 2
- 150000001447 alkali salts Chemical class 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000002518 antifoaming agent Substances 0.000 description 2
- 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 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 210000003850 cellular structure Anatomy 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 238000005189 flocculation Methods 0.000 description 2
- 230000016615 flocculation Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 235000012054 meals Nutrition 0.000 description 2
- 239000012533 medium component Substances 0.000 description 2
- QMPFMODFBNEYJH-UHFFFAOYSA-N methyl 1h-1,2,4-triazole-5-carboxylate Chemical compound COC(=O)C1=NC=NN1 QMPFMODFBNEYJH-UHFFFAOYSA-N 0.000 description 2
- 235000013379 molasses Nutrition 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 150000003014 phosphoric acid esters Chemical class 0.000 description 2
- 238000011085 pressure filtration Methods 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- 238000003828 vacuum filtration Methods 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- RQFCJASXJCIDSX-UHFFFAOYSA-N 14C-Guanosin-5'-monophosphat Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(O)=O)C(O)C1O RQFCJASXJCIDSX-UHFFFAOYSA-N 0.000 description 1
- JDIIGWSSTNUWGK-UHFFFAOYSA-N 1h-imidazol-3-ium;chloride Chemical compound [Cl-].[NH2+]1C=CN=C1 JDIIGWSSTNUWGK-UHFFFAOYSA-N 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- XNWFRZJHXBZDAG-UHFFFAOYSA-N 2-METHOXYETHANOL Chemical compound COCCO XNWFRZJHXBZDAG-UHFFFAOYSA-N 0.000 description 1
- 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 1
- ZNQVEEAIQZEUHB-UHFFFAOYSA-N 2-ethoxyethanol Chemical compound CCOCCO ZNQVEEAIQZEUHB-UHFFFAOYSA-N 0.000 description 1
- 229940093475 2-ethoxyethanol Drugs 0.000 description 1
- DCTLYFZHFGENCW-UUOKFMHZSA-N 5'-xanthylic acid Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(NC(=O)NC2=O)=C2N=C1 DCTLYFZHFGENCW-UUOKFMHZSA-N 0.000 description 1
- 241000590020 Achromobacter Species 0.000 description 1
- 241000607534 Aeromonas Species 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 241000186063 Arthrobacter Species 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000186146 Brevibacterium Species 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 1
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
〔〕 発明の背景
技術分野
本発明はリバビリン（Ribavirin）の微生物を
利用する製造法に関するものである。
リバビリンの化学名は１−β−Ｄ−リボフラノ
シル−１，２，４−トリアゾール−３−カルボキ
サミドであり、バイラゾール（Virazole）とも
称され、DNAおよびRNAウイルスに対して広範
囲で強力な抗ウイルス作用を示す化合物として知
られている（アナルズ・オブ・ザ・ニユーヨー
ク・アカデミー・オブ・サイエンシズ（Ann.
New York Acad.Sci.）284、272〜292（1977））。
従来技術
従来知られているリバビリンの製造法としては
合成法と微生物の培養による方法と酵素的な方法
がある。
合成法の代表的な方法としては、３−メトキシ
カルボニル−１，２，４−トリアゾールと１−Ｏ
−アセチル−２，３，５−トリ−Ｏ−アシル−β
−Ｄ−リボフラノースを反応させ（溶融法）、得
られた１−（2′，3′，5′−トリ−Ｏ−アシル−β−
Ｄ−リボフラノシル）−３−メトキシカルボニル
−１，２，４−トリアゾールをアンモニアで処理
し、アミド化と脱保護を行う方法（特開昭48−
4469号、特開昭49−80070号、特開昭49−80071号
各公報参照）、前記と同様の方法においてトリア
ゾールの３位の置換基としてアラルキルオキシ基
を用いる方法（特開昭55−160793号公報参照）、
３−メトキシカルボニル−１，２，４−トリアゾ
ールをトリメチルシリル化し、２，３，５−トリ
−Ｏ−ベンゾイル−β−Ｄ−リボフラノシドのハ
ロゲン化物と反応させた（シリル化法）後、アン
モニアで処理する方法（特開昭48−4469号、特開
昭49−86372号各公報参照）などが知られている。
このような合成法は、いずれも反応前に原料化合
物の活性基を保護する必要があり、また反応に際
してはリボースの活性化が必要であつたり、高温
に加熱する必要がある場合もあり、さらに、反応
後に脱保護およびアミド化が必要であるなど反応
操作が煩雑であるなどの問題がある。また、縮合
反応の位置選択性はいずれも高くない。
微生物の培養による方法としては、ブレビバク
テリウム属、コリネバクテリウム属、アースロバ
クター属、ミクロコツカス属またはバチルス属に
属する微生物を培養して増殖させる際に、使用微
生物の培養のために必要な炭素源、窒素源、無機
物、その他の栄養物を含有する培地に、培養開始
前または培養中、一時にまたは間歇的に１，２，
４−トリアゾール−３−カルボキサミド（以下、
「トリアゾール化合物」と略すこともある。）を添
加し、培養開始後２〜８日間という長期間にわた
つて培養し、培地中にリバビリンを生成蓄積せし
める方法が知られている（特公昭54−17830号公
報、日本農芸化学会誌、50(9)、423〜430（1976）
参照）。本発明とこの方法とは、使用する微生物
の種類において相異し、その効果も格段に異るも
のである。
また、酵素的な製造法としては１，２，４−ト
リアゾール−３−カルボキサミドとリボース−１
−りん酸とをPH５〜９、温度０〜50℃の条件下で
ヌクレオシドホスホリラーゼの存在下において反
応させる方法が知られている（特開昭50−29720
号公報参照）。この方法においてはリボース供与
体としてリボース−１−りん酸が開示されている
にすぎない。また、酵素源として動物または微生
物より得られるヌクレオシドホスホリラーゼを使
用できる旨の記載があり、微生物としてエシエリ
ヒア・コリおよび酵母が例示されているが、具体
的には子牛脾臓由来のヌクレオシドホスホリラー
ゼを用いる方法が示されているにすぎず、微生物
の酵素については「活発に代謝するバクテリア又
は真菌細胞中に存在」する酵素をこれらの微生物
を培養することによつて利用しうる可能性が示唆
されているにすぎない。本発明の方法は、リボー
ス供与体としてヌクレオチド、ヌクレオチドなど
から広く選択できること、使用微生物も各種のも
のを選択できること、ならびに使用微生物の培養
物、菌体または菌体処理物をその非増殖条件下、
たとえば高温度条件下において酵素源として反応
に使用することができることなどの点において前
記の方法とは全く異るものである。
〔〕 発明の概要
要 旨
本発明者らは、トリアゾール化合物とリボース
供与体に作用し、リバビリンを生成する能力を有
する微生物が細菌および酵母の間に多種類分布し
ていることを知見し、この知見に基づいて本発明
を完成した。
本発明は、シユウドモナス属、スタフイロコツ
カス属、フラボバクテリウム属、エシエリヒア
属、セルロモナス属、エンテロバクター属、ビフ
リオ属、エルヴイニア属またはクレブシエラ属に
属し、１，２，４−トリアゾール−３−カルボキ
サミドとリボース供与体であるヌクレオチドとか
らリバビリンを生成する能力を有する微生物の作
用により、１，２，４−トリアゾール−３−カル
ボキサミドまたはその塩およびヌクレオチドまた
はその塩を水性媒体中で反応させてリバビリンを
生成させ、これを取得することを特徴とするリバ
ビリンの製造法を提供するものである。
すなわち、本発明は前記の微生物の培養物、菌
体または菌体処理物（以下、これらを総括的に
「菌体等」と称することもある。）を酵素源とし、
トリアゾール化合物およびリボース供与体にこの
ような菌体等を接触させることによつて作用さ
せ、リバビリンを製造することを目的とするもの
である。
効 果
本発明においては微生物の非増殖条件下におい
て、微生物の培養物、菌体または菌体処理物を酵
素源とし、酵素反応に必要な基質のみを含有する
反応液中で反応を行うこともできる。このような
方法による場合には基質溶液の調製が簡単であ
り、反応時間は培養法に比べて短く、副生物の生
成もほとんどなく、反応液からリバビリンの分離
精製が容易であり、菌体等の反復または連続使用
も可能であるなどの利点を有する。また、非増殖
条件下における酵素反応法として高温条件下で反
応を行う場合には、反応速度が増大し、反応液の
雑菌汚染がほとんどないだけでなく、合成された
リバビリンの分解反応も抑制されるなどの効果が
ある。
〔〕 発明の具体的説明
使用微生物
本発明において使用される微生物は、トリアゾ
ール化合物とリボース供与体に作用してリバビリ
ンを生成する能力を有するものであり、広く細
菌、酵母などに分布するものである。具体的には
フラボバクテリウム（Flavobacterium；以下、
「F.」と略す。）属、ミクロバクテリウム
（Microbacterium；以下、「M.」と略す。）属、
キサントモナス（Xanthomonas；以下、「X.」
と略す。）属、シユウドモナス（Pseudomonas；
以下、「Ps.」と略す。）属、アクロモバクター
（Achromobacter；以下、「Ach.」と略す。）属、
エシエリヒア（Escherichia；以下、「E.」と略
す。）属、エアロバクター（Aerobacter；以下、
「Aer.」と略す。）属、サルシナ（Sarcina；以
下、「S.」と略す。）属、スタフイロコツカス
（Staphylococcus；以下、「Sta.」と略す。）属、
バクテリウム（Bacterium；以下、「B.」と略
す。）属、セラチア（Serratia；以下、「Ser.」と
略す。）属、プロテウス（Proteus；以下、「Pro.」
と略す。）属、セルロモナス（Cellulomonas；以
下、「C.」と略す。）属、エンテロバクター
（Enterobacter；以下、「Ent.」と略す。）属、ミ
コプラナ（Mycoplana；以下、「Myco.」と略
す。）属、ビブリオ（Vibrio；以下、「V.」と略
す。）属、エルヴイニア（Erwinia；以下、「Er.」
と略す。）属、クレブシエラ（Klebsiella；以下、
「K.」と略す。）属、エアロモナス
（Aeromonas；以下、「Aermo.」と略す。）属、
ミコトルラ（Mycotorula；以下、「Myct.」と略
す。）属またはキヤンデイダ（Candida；以下、
「Can.」と略す。）属に属し、前記の作用を有す
る微生物が挙げられる。本発明においてはこのよ
うな基本的性質を有するものである限り、特に使
用菌株の種類に限定されるものではない。以下に
当業者が容易に入手できる公知の菌株を例示す
る。
[] BACKGROUND TECHNICAL FIELD OF THE INVENTION The present invention relates to a method for producing ribavirin using microorganisms. The chemical name of ribavirin is 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, also known as Virazole, which has a broad range of potent antiviral effects against DNA and RNA viruses. (Annals of the New York Academy of Sciences (Ann.
New York Acad. Sci.) 284 , 272-292 (1977)). Prior Art Conventionally known methods for producing ribavirin include a synthetic method, a method by culturing microorganisms, and an enzymatic method. A typical synthesis method is to use 3-methoxycarbonyl-1,2,4-triazole and 1-O
-acetyl-2,3,5-tri-O-acyl-β
-D-ribofuranose (melting method) and the obtained 1-(2',3',5'-tri-O-acyl-β-
D-ribofuranosyl)-3-methoxycarbonyl-1,2,4-triazole is treated with ammonia to amidate and deprotect
4469, JP-A-49-80070, and JP-A-49-80071), a method using an aralkyloxy group as a substituent at the 3-position of the triazole in the same method as above (JP-A-55-160793) (see publication),
3-Methoxycarbonyl-1,2,4-triazole was trimethylsilylated and reacted with a halide of 2,3,5-tri-O-benzoyl-β-D-ribofuranoside (silylation method), and then treated with ammonia. There are known methods to do this (see Japanese Patent Laid-Open Nos. 48-4469 and 86372-1987).
In all of these synthetic methods, it is necessary to protect the active groups of the raw material compounds before the reaction, and during the reaction, it may be necessary to activate ribose, or it may be necessary to heat to high temperatures. However, there are problems such as complicated reaction operations such as the need for deprotection and amidation after the reaction. Furthermore, the regioselectivity of the condensation reaction is not high. The method of culturing microorganisms involves culturing and propagating microorganisms belonging to the genus Brevibacterium, Corynebacterium, Arthrobacter, Micrococcus, or Bacillus. Add 1, 2, 1, 2, 1, 2, 1, 2,
4-triazole-3-carboxamide (hereinafter referred to as
Sometimes abbreviated as "triazole compound". ) and culturing for a long period of 2 to 8 days after the start of culture to produce and accumulate ribavirin in the medium (Special Publication No. 17830/1983, Journal of the Japanese Society of Agricultural Chemistry, 50) . (9), 423-430 (1976)
reference). The present invention and this method differ in the type of microorganisms used, and their effects are also significantly different. In addition, as an enzymatic production method, 1,2,4-triazole-3-carboxamide and ribose-1
A method is known in which phosphoric acid is reacted with phosphoric acid in the presence of nucleoside phosphorylase at a pH of 5 to 9 and a temperature of 0 to 50°C.
(see publication). In this method, only ribose-1-phosphate is disclosed as the ribose donor. Furthermore, it is stated that nucleoside phosphorylase obtained from animals or microorganisms can be used as an enzyme source, and Escherichia coli and yeast are exemplified as microorganisms, but specifically, a method using nucleoside phosphorylase derived from calf spleen is described. However, it has been suggested that enzymes from microorganisms that are "present in actively metabolizing bacterial or fungal cells" may be utilized by culturing these microorganisms. It's nothing more than that. In the method of the present invention, the ribose donor can be selected from a wide variety of nucleotides, nucleotides, etc., various types of microorganisms can be used, and the culture, cells, or treated microorganisms of the microorganism used can be used under non-proliferation conditions.
This method is completely different from the above method in that, for example, it can be used as an enzyme source in reactions under high temperature conditions. [] Summary of the Invention The present inventors have discovered that there are many types of microorganisms distributed among bacteria and yeast that have the ability to act on triazole compounds and ribose donors to produce ribavirin. The present invention was completed based on this knowledge. The present invention relates to a 1,2,4-triazole-3-carboxamide which belongs to the genus Pseudomonas, Staphylococcus, Flavobacterium, Escherichia, Cellulomonas, Enterobacter, Bifrio, Erwinia or Klebsiella. Ribavirin is produced by reacting 1,2,4-triazole-3-carboxamide or its salt and nucleotide or its salt in an aqueous medium by the action of a microorganism that has the ability to produce ribavirin from The present invention provides a method for producing ribavirin, which is characterized by producing and obtaining ribavirin. That is, the present invention uses the above-mentioned microorganism culture, bacterial cells, or treated bacterial cells (hereinafter, these may be collectively referred to as "microbial cells, etc.") as an enzyme source,
The purpose of this method is to produce ribavirin by bringing such microbial cells into contact with a triazole compound and a ribose donor. Effects In the present invention, it is also possible to carry out the reaction under conditions in which microorganisms do not proliferate, using microorganism cultures, microbial cells, or processed microbial cells as enzyme sources, and in a reaction solution containing only the substrates necessary for the enzymatic reaction. can. When using this method, the preparation of the substrate solution is easy, the reaction time is shorter than that of the culture method, there is almost no production of by-products, it is easy to separate and purify ribavirin from the reaction solution, and bacterial cells etc. It has the advantage that it can be used repeatedly or continuously. In addition, when the reaction is carried out under high temperature conditions as an enzymatic reaction method under non-proliferation conditions, the reaction rate not only increases and there is almost no bacterial contamination of the reaction solution, but also the decomposition reaction of the synthesized ribavirin is suppressed. It has the effect of [] Specific description of the invention Microorganism used The microorganism used in the present invention has the ability to produce ribavirin by acting on a triazole compound and a ribose donor, and is widely distributed among bacteria, yeast, etc. . Specifically, Flavobacterium (hereinafter referred to as
Abbreviated as "F." ) genus, Microbacterium (hereinafter abbreviated as "M.") genus,
Xanthomonas (hereinafter referred to as "X.")
It is abbreviated as ), Pseudomonas;
Hereinafter, abbreviated as "Ps." ) genus, Achromobacter (hereinafter abbreviated as "Ach.") genus,
Escherichia (hereinafter abbreviated as "E.") genus, Aerobacter (hereinafter abbreviated as "E."),
Abbreviated as "Aer." ) genus, Sarcina (hereinafter abbreviated as "S.") genus, Staphylococcus (hereinafter abbreviated as "Sta.") genus,
Bacterium (hereinafter abbreviated as "B.") genus, Serratia (hereinafter abbreviated as "Ser.") genus, Proteus (hereinafter abbreviated as "Pro.")
It is abbreviated as ) genus, Cellulomonas (hereinafter abbreviated as "C.") genus, Enterobacter (hereinafter abbreviated as "Ent.") genus, Mycoplana (hereinafter abbreviated as "Myco."). Genus, Vibrio (hereinafter abbreviated as "V.") Genus, Erwinia (hereinafter "Er.")
It is abbreviated as ) genus, Klebsiella (hereinafter referred to as
Abbreviated as "K." ) genus, Aeromonas (hereinafter abbreviated as "Aermo.") genus,
Genus Mycotorula (hereinafter abbreviated as "Myct.") or Candida (hereinafter referred to as "Myct.")
Abbreviated as "Can." ) belonging to the genus and having the above-mentioned effects. In the present invention, the type of strain used is not particularly limited as long as it has these basic properties. Examples of known strains that are easily available to those skilled in the art are listed below.
【表】
上記菌株は、工業技術院微生物工業技術研究所
（以下、「微工研」と略称する。）に対し、昭和56
年通商産業省告示による寄託（以下、国内寄託と
称する。）の申請を行い、昭和57年５月24日付け
で受託され、受託番号として微工研菌寄第6538号
（FERM Ｐ−6538）が付与されている。[Table] The above bacterial strains were submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology (hereinafter referred to as "Feikoken") in 1982.
An application was made for deposit (hereinafter referred to as domestic deposit) in accordance with the notification of the Ministry of International Trade and Industry in 1983, and the deposit was received on May 24, 1981, with the deposit number being FERM P-6538. has been granted.
【表】
上記菌株は、微工研に対して国内寄託され、昭
和57年５月24日付けで微工研菌寄第6539号
（FERM Ｐ−6539）が付与されている。
バクテリウム・サクシニカム
（B.succinicum） IAM1017
上記菌株は、微工研に対して国内寄託され、昭
和57年５月24日付けで微工研菌寄第6540号
（FERM Ｐ−6540）が付与されている。[Table] The above strain has been deposited domestically with the FERM, and has been assigned FERM P-6539 on May 24, 1981. B. succinicum (B. succinicum) IAM1017 The above strain has been deposited domestically with the FERM, and has been assigned FERM Bacteria No. 6540 (FERM P-6540) on May 24, 1981. There is.
【表】
上記菌株は、微工研に対して国内寄託され、昭
和57年５月24日付けで微工研菌寄第6541号
（FERM Ｐ−6541）が付与されている。
なお、上記菌株の寄託番号において、ATCCを
付した番号はアメリカン・タイプカルチユアー・
コレクシヨン（The American Type Culture
Collection）における寄託番号を、IFOを付した
番号は財団法人 発酵研究所（Institute for
Fermentation、Osaka）における寄託番号を、
IAMを付した番号は東京大学応用微生物研究所
（Institute of Applied Microbiology、
University of Tokyo）における寄託番号を、
OUTを付した番号は大阪大学工学部（Faculty
of Engineering、Osaka University）における
寄託番号をそれぞれ示すものであり、これらの菌
株は当該寄託機関の保存菌株リストに収載されて
いる保存菌である。またIFO、OUTおよびIAM
番号を付された菌株は日本微生物保存株目録
（JFCC Catalogue of Cultures）に収載されて
いる保存菌株である。これらの保存菌株の属名ま
たは種名は分類基準の変更などによつて変ること
もあるが、このような菌株であつても前記例示の
菌株と同一もしくは均等の菌学的性質を有するも
のは、その属する属名にかかわらず前記例示の菌
株と同一とみなされる。
また、前記の菌株から、紫外線、Ｘ線、γ線の
照射などの物理的処理もしくはニトロソグアニジ
ンなどによる薬剤処理など、一般的変異誘導法に
よる誘発突然変異または自然の原因に起因する自
然突然変異によつて誘導された変異株も、本発明
の目的とするリバビリン製造に関与する酵素活性
を失なわない限り、本発明に使用される。
さらに、以上のような本発明に好適に使用され
る菌株から得られた本発明の目的とするリバビリ
ン製造に関与する酵素系の遺伝子が前記例示の属
以外の微生物に取り込まれ、そのような形質が発
現するに至つた場合、このような微生物を本発明
の目的に使用する方法は、本発明に包含される。
培 養
本発明に使用される菌体等を得るために前記の
微生物を培養するに際し、使用される培地および
培養法は、これらの微生物が生育する限り、特に
限定されない。
培地としてはこれらの微生物が資化可能な炭素
源および窒素源を適当量含有し、必要に応じて無
機塩、微量発育促進物質、消泡剤などを添加した
ものが使用される。具体的には、炭素源として
は、グルコース、フラクトース、マルトース、ガ
ラクトース、リボース、シユークロース、澱粉、
澱粉加水分解物、糖蜜、廃糖蜜などの糖類もしく
はその脂肪酸エステルなどの誘導体、麦、麦芽エ
キス、〓、米などの天然炭水化物、グリセロー
ル、マンニトール、メタノール、エタノールなど
のアルコール類、グルコン類、ピルビン酸、酢
酸、クエン酸などの脂肪酸類、ノルマルパラフイ
ン、ケロシンなどの炭化水素類、グリシン、グル
タミン酸、グルタミン、アラニン、アスパラギン
などのアミノ酸類など、一般的な炭素源より使用
する微生物の資化性を考慮して一種または二種以
上を適宜に選択して使用すればよい。窒素源とし
ては、肉エキス、ペプトン、酵母エキス、乾燥酵
母、大豆加水分解物、大豆粉、ミルクカゼイン、
カザミノ酸、各種アミノ酸、コーンステイープリ
カー、コツトンシードミールないしその加水分解
物、フイツシユミールないしその加水分解物、そ
の他の動物、植物、微生物の加水分解物などの有
機窒素化合物、アンモニア、硝酸アンモニウム、
硫酸アンモニウム、塩化アンモニウム、りん酸ア
ンモニウム、炭酸アンモニウム、酢酸アンモニウ
ムなどのアンモニウム塩、硝酸ナトリウムなどの
硝酸塩、尿素など無機窒素化合物より使用微生物
の資化性を考慮し、一種または二種以上を適宜に
選択して使用する。さらに、無機塩としてマグネ
シウム、マンガン、鉄、亜鉛、銅、ナトリウム、
カルシウム、カリウムなどのりん酸塩、塩酸塩、
硝酸塩、炭酸塩、硝酸塩、酢酸塩などの一種また
は二種以上を適宜添加し、必要に応じて植物油、
界面活性剤などの消泡剤、ビタミンB1、B2、ニ
コチン酸、パントテン酸、ビオチン、ｐ−アミノ
安息香酸などの微量発育促進物質を添加してもよ
い。また、栄養要求を同時に示す微生物を使用す
る場合、当然その生育を満足させる物質を培地に
添加しなければならない。
培養の方法は特に限定されないが、通常前記培
地成分を含有する液体培地中で振盪培養、通気撹
拌培養、静置培養、連続培養などの通常の培養法
より使用微生物に適した培養法を選択して行う。
培養条件は、使用微生物および培地の種類によ
り適宜選択すればよいが、菌体等の取得を目的と
する場合、通常は培養開始のPHを約６〜８に調整
し、約25〜35℃の温度条件下で培養を行う。培養
期間は使用微生物の生育に十分な時間であればよ
く、通常１〜３日間である。
なお、以上のような菌体等を得るための微生物
の培養の際に、培地中にトリアゾール化合物およ
びリボース供与体を存在させて、これらの反応基
質に微生物を作用させてもよい。
酵素源
本発明の方法を実施するに際しては前記の菌体
等を酵素源として使用し、これと反応基質溶液を
接触させて反応を行うが、以下にこのような酵素
源の使用形態および製造法を示す。
すなわち、以上のように微生物を培養し、得ら
れた培養物、培養物から遠心分離、沈降分離、凝
集分離、濾過（加圧濾過、真空濾過などを含む。）
などの通常の方法によつて集菌した生菌体、また
は生菌体に適宜な処理を施して得られる菌体処理
物を本発明における酵素源として使用できる。こ
こで、培養物とは培養後の培地と培養菌体が未分
離の状態のものをいう。また、菌体処理物とは、
乾燥菌体、細胞膜・壁変性菌体、破砕菌体、固定
化菌体、菌体抽出物、本発明の目的とするリバビ
リンの製造に関与する酵素活性を有する菌体抽出
物の蛋白質画分もしくはその精製物、蛋白質画分
もしくはその精製物の固定化物などを指称する。
菌体処理物を得るための方法を以下に例示す
る。すなわち、生菌体に対し、たとえば凍結融
解処理、凍結乾燥処理、通風乾燥処理、アセトン
乾燥処理、酸性ないしアルカリ性下における加温
処理、磨砕処理、超音波処理、浸透圧差処理など
の物理的処理手段、もしくはたとえば、リゾチー
ム、細胞壁溶解酵素などの酵素処理、トルエン、
キシレン、ブチルアルコール（ブタノール）など
の溶媒もしくは界面活性剤との接触処理などの化
学的ないし生物化学的処理を単独もしくは組み合
せて施すことにより、また、菌体抽出物に対
し、たとえば塩析処理、等電点沈澱処理、有機溶
媒沈澱処理、各種クロマトグラフ処理、透析処理
などの酵素分離精製手段を単独もしくは組み合せ
て施すことにより、さらに、生菌体、菌体抽出
物もしくはその精製物に包括処理、架橋処理、担
体への吸着処理などの酵素固定化手段を単独もし
くは適宜に組み合せて施すことにより菌体処理物
を得ることができる。
反応基質
本発明の酵素反応における反応基質は１，２，
４−トリアゾール−３−カルボキサミドおよびリ
ボース供与体である。
１，２，４−トリアゾール−３−カルボキサミ
ドは遊離型またはナトリウム塩などのアルカリ塩
もしくは酸付加塩のいずれも使用できる。
リボース供与体としてはリボヌクレオシドもし
くはＤ−リボースまたはこれらの各種りん酸エス
テルなどのリボース誘導体であればいずれも使用
できる。さらに、このようなリボース誘導体以外
の糖類であつて、使用する微生物に代謝されてリ
ボース誘導体に導かれうるものは本発明において
リボース供与体とみなされる。なお、これらのリ
ボース供与体は必らずしも反応基質溶液中に添加
する必要はなく、菌体等の中にその本来の成分と
して含有されているものを利用してもよい。ま
た、このような菌体成分としてのリボース供与体
が酵素反応によつて消費されて減少した場合には
系外から添加すればよい。
系外より添加される好適なリボース供与体を以
下に例示する。すなわち、リボヌクレオシドはそ
の塩基部分がプリン系またはピリミジン系のいか
なる塩基であつてもよく、天然物由来であれ化学
合成によるものであれ使用することができる。ま
た、リボヌクレオシドもしくはＤ−リボースの糖
部水酸基は非置換のものであつてもあるいは２
位、３位もしくは５位水酸基のいずれか一個所、
二個所もしくは全てがモノりん酸エステル、ジり
ん酸エステル、トリりん酸エステルのようなりん
酸エステル化されたものであつてもよい。また、
これらのりん酸エステルは遊離型であつてもよ
く、またナトリウム、カリウム、カルシウム、マ
グネシウム、アンモニウム、トリエチルアンモニ
ウムなどの一般的なアルカリ塩であつてもよい。
リボース供与体の具体例としてはイノシン、アデ
ノシン、グアノシン、キサントシン、ウリジン、
シチジン、5′−イノシン酸、5′−アデニル酸、
5′−グアニル酸、5′−キサンチル酸、5′−ウリジ
ル酸、5′−シチジル酸、2′（3′）−イノシン酸、
2′（3′）−アデニル酸、2′（3′）−グアニル酸、2
′（3′）
−キサンチル酸、2′（3′）−ウリジル酸、2′（3′）
−
シチジル酸、Ｄ−リボース、Ｄ−リボース−１−
りん酸などが例示される。また、リボース誘導体
に代謝されうる糖類としてはグルコース、シユー
クロースなどが例示される。
反応基質溶液
本発明の酵素反応に使用される基質溶液は、基
本的には前記の反応基質が水性媒体に溶解もしく
は懸濁した水性液である。
水性液中には少なくとも前記のトリアゾール化
合物の一種または二種以上および前記のリボース
供与体の一種または二種以上を含有し、これらの
反応基質のほかに、りん酸イオン供与系、有機溶
媒、界面活性剤、金属塩類、補酵素類、酸、塩
基、糖類など酵素反応を促進する物質、妨害酵素
活性を阻害する物質、反応基質の溶解性を向上さ
せる物質、酵素と反応基質の接触を向上させる物
質等を含有していてもよい。また、使用微生物が
資化しうる前記のような培地成分を含有していて
もよい。
水性媒体としては、水または酵素反応に好適な
各種緩衝液（りん酸緩衝液、イミダゾール−塩酸
緩衝液、ベロナール−塩酸緩衝液、トリス−塩酸
緩衝液など）を用いることができる。
りん酸イオン供与系としては、水性媒体中でり
ん酸イオンに解離しうるもののいずれを用いても
よく、たとえば遊離型りん酸そのもの、無機りん
酸塩、たとえばナトリウム、カリウムなどのアル
カリ金属、カルシウム、マグネシウムなどのアル
カリ土類金属、アンモニウムとの塩が好適に使用
される。また、りん酸イオン供与系としては、酵
素反応の基質溶液中でりん酸イオンを遊離しうる
系、たとえば各種りん酸エステル誘導体とホスフ
アターゼの組み合せ、ヌクレオチドとヌクレオチ
ダーゼの組み合せ、核酸塩基およびリボース−１
−りん酸とホスホリラーゼの組み合せなどを利用
することができる。
以上のようなりん酸供与系は酵素反応に際して
系外から添加されたものであつてもよく、使用微
生物の成分として含有されているものであつても
よい。すなわち、酵素反応に利用しうる形態であ
る限り、上記の物質の単独もしくは二種以上を租
み合せた系を、または上記の物質を含有する微生
物菌体もしくはその菌体処理物を本発明の酵素反
応に際して反応液に別途添加してもよく、使用微
生物の菌体成分として含有されているこれらの物
質をそのまま利用してもよい。
有機溶媒としては、たとえばメタノール、エタ
ノール、プロパノール、ブタノール、ペンタノー
ル、アセトン、メチルエチルケトン、酢酸エチ
ル、トルエン、テトラヒドロフラン、ジオキサ
ン、ジメチルスルホキシド、ジメチルアセトアミ
ド、ジメチルホルムアミド、２−メトキシエタノ
ール、２−エトキシエタノール、１，２−ジメト
キシエタンなどが例示される。
接触方法
本発明の反応は、前記の酵素源と反応基質とを
水性媒体中で接触させることにより達成される。
接触方法は、酵素源の形態に応じて適宜に選択
すればよいが、通常、酵素源を反応基質溶液に懸
濁もしくは溶解し、好ましくは加温しながら攪拌
もしくは振盪するバツチ方式、または酵素源を必
要に応じて適当な担体、助剤、吸着剤と混和し、
もしくはこれらに担持させてカラムに充填し、反
応基質溶液を通液するカラム方式などが適用され
る。なお、バツチ方式の場合には反応後、菌体等
を濾過（加圧濾過、真空濾過などを含む。）、遠心
分離、沈降分離、凝集分離など通常の方法によつ
て集菌し、反応基質溶液と接触させることによつ
て繰り返し使用することができる。カラム方式の
場合は、菌体等の分離操作は必要ないが、同様に
繰り返し、もしくは連続的に酵素反応に使用する
ことができる。また、使用微生物の培養に際し、
培地中に反応基質を添加し、酵素源と反応基質を
反応させる方法も本発明に採用しうる。
反応基質および酵素源の濃度もしくは添加量
反応に際し、反応液の基質濃度は特に制限され
るものではなく、反応温度における使用水性媒体
に対する各基質の飽和濃度以下の基質濃度が通常
採用されるが、反応基質溶液に添加された前記の
有機溶媒、界面活性剤などにより基質濃度を増大
させることもできる。また、反応液中に飽和濃度
以上の各基質を懸濁状態で存在させ、反応の進行
に従つて各基質を溶解させることもできる。ま
た、各基質を反応中に逐次添加し、適当濃度に保
つこともできる。各基質を添加し、溶解させる場
合、基質濃度は１，２，４−トリアゾール−３−
カルボキサミドまたはその塩については通常５〜
200ｍＭ程度、好ましくは10〜100ｍＭ程度であ
り、リボース供与体については通常０〜300ｍＭ
程度、好ましくは１〜150ｍＭ程度である。
酵素源の使用量は微生物の種類、その使用形
態、反応効率、経済性などを考慮し、当業者が予
備実験等によつて容易に決定できるものである
が、通常バツチ方式の場合、たとえば生（湿）菌
体であれば10〜150mg／ml基質溶液程度、乾燥菌
体であれば２〜30mg／ml基質溶液程度であればよ
く、カラム方式においてはバツチ方式に準じて適
当な量を設定することができる。
反応条件
本発明の反応の条件は、反応基質が菌体等の作
用によつて反応し、効率よくリバビリンが生成す
る条件であれば使用微生物の非増殖条件下であれ
増殖条件下であれ特に限定されない。しかしなが
ら、使用微生物の非増殖条件下における反応が特
に効率が良い。
微生物の非増殖条件下で反応に供する方法とし
ては、酵素反応温度を使用微生物が増殖できない
温度範囲（ただし、本発明の反応に関与する酵素
が失活しない温度範囲である。）に設定する方法、
使用微生物菌体をあらかじめ前記のとおり物理
的、化学的ないし生物化学的に処理することによ
つて微生物を増殖できない状態にした後、反応に
供する方法、反応に際して、たとえばトルエンな
どの使用微生物の増殖を阻害する物質を反応基質
溶液に添加する方法などを単独にあるいは組み合
せて採用すればよいが、特に反応温度を操作する
方法が最も効果的で簡便である。
本発明の反応は28〜80℃の範囲において進行す
るが、実用性を考慮すれば37〜70℃の範囲が好ま
しく、特に45〜60℃が最適である。なお、37℃以
上の温度においては、反応によつて生成したリバ
ビリンが反応液中に存在する菌体等の作用によつ
て分解する反応が抑制されるのでリバビリンの生
成効率が良い。
反応基質溶液の液性は、通常PH４〜10、好まし
くはPH６〜８の範囲に保たれればよく、反応中に
PHが変動するときは、塩酸、硫酸、りん酸などの
酸または水酸化ナトリウム、水酸化カリウム、ア
ンモニア水、アンモニアガスなどのアルカリを用
いて好ましいPH範囲に補正すればよい。
反応時間は、反応基質の目的物への変換率を確
認しながら決定すればよいが、通常バツチ方式で
は２〜45時間程度、好ましくは24〜36時間程度反
応させればよく、カラム方式ではバツチ方式に準
じて適当な条件を設定して反応させればよい。
分離精製
反応後、必要に応じて菌体等を濾過、遠心分離
または凝集分離などの常法によつて分離除去し、
リバビリンの分離精製工程に供する。
リバビリンの分離精製は、公知の方法またはこ
れを応用して行えばよく、たとえばイオン変換ク
ロマトグラフイー、吸着クロマトグラフイー、分
配クロマトグラフイー、ゲル濾過法など各種のク
ロマトグラフイー、向流分配、向流抽出など二液
相間の分配を利用する方法、濃縮、冷却、有機溶
媒添加など溶解度の差を利用する方法などの一般
的な分離精製法を単独で、あるいは適宜に組み合
せて行えばよい。
分 析
本発明の実施例においてリバビリンおよび１，
２，４−トリアゾール−３−カルボキサミドの分
析は高速液体クロマトグラフイーによつて行つ
た。以下に示す装置および条件で分析すると、リ
バビリンは保持時間3.50分付近に、１，２，４−
トリアゾール−３−カルボキサミドは保持時間
2.65分付近に溶出され、検量線よりそれぞれの量
を算出できる。
装置：島津高速液体クロマトグラフLC−3A型
（(株)島津製作所製）
カラム：マイクロ・ボンダパツク
（μBONDAPAK）C18、4.6mm×250mm（日本ウ
オーターズリミテツド社製）
溶出剤：２％アセトニトリルを含む20ｍＭトリス
一塩酸緩衝液（PH7.5）
流速：１ml／分
測定波長：225nｍ
カラム操作温度：室温
〔〕 実施例
以下、実施例をもつて本発明をより具体的に説
明するが、これらはいずれも実施の一態様を示す
ものであつて、本発明の範囲を制限するものでは
ない。
実施例 １
第１表に示す各菌株を粉末ブイヨン（極東製薬
工業(株)製）２％水溶液各50mlに植菌し、28℃、24
時間振盪培養後、遠心分離によつて集菌し、殺菌
水を加えて菌体懸濁液各５mlを得た。
20ｍM1，２，４−トリアゾール−３−カルボ
キサミド、20ｍＭイノシンおよび25ｍＭりん酸−
カリウムを含む水溶液（PH7.0）各５mlに前記菌
体懸濁液各５mlを加え、60℃、20時間反応した。
反応終了後、遠心分離によつて菌体を除去し、リ
バビリン生成率を分析したところ第１表に示すと
おりであつた。なお、リバビリン生成率とは１，
２，４−トリアゾール−３−カルボキサミドから
リバビリンへの転換率（％）をいう。[Table] The above strain has been deposited domestically with the FERM, and has been assigned FERM Bacteria No. 6541 (FERM P-6541) dated May 24, 1981. In addition, in the deposit number of the above strain, the number with ATCC is American Type Culture.
Collection (The American Type Culture
The deposit number in the Fermentation Research Institute (IFO Collection) is the number with IFO.
Fermentation, Osaka) deposit number.
Numbers with IAM are from Institute of Applied Microbiology, University of Tokyo.
University of Tokyo) deposit number,
Numbers with OUT are for Osaka University Faculty of Engineering.
of Engineering, Osaka University), and these strains are stored in the list of stored strains of the depositary institution. Also IFO, OUT and IAM
The numbered strains are stored strains listed in the Japan FCC Catalog of Cultures. The genus or species names of these stored strains may change due to changes in classification standards, but even such strains may have the same or equivalent mycological properties as the above-mentioned strains. , are considered to be the same as the above-mentioned exemplified strains, regardless of the genus name to which they belong. In addition, the above-mentioned strains may undergo induced mutations by general mutagenesis methods such as physical treatments such as irradiation with ultraviolet rays, The mutant strain thus derived can also be used in the present invention, as long as it does not lose the enzymatic activity involved in ribavirin production, which is the object of the present invention. Furthermore, genes for enzyme systems involved in ribavirin production, which is the object of the present invention, obtained from the strains preferably used in the present invention as described above are incorporated into microorganisms other than the above-mentioned genera, and such traits are obtained. If such a microorganism is expressed, the method of using such a microorganism for the purpose of the present invention is encompassed by the present invention. Culture When culturing the above-mentioned microorganisms in order to obtain the microorganisms used in the present invention, the medium and culture method used are not particularly limited as long as these microorganisms can grow. The medium used contains appropriate amounts of carbon sources and nitrogen sources that can be assimilated by these microorganisms, and if necessary, inorganic salts, trace growth-promoting substances, antifoaming agents, etc. are added. Specifically, carbon sources include glucose, fructose, maltose, galactose, ribose, sucrose, starch,
Starch hydrolysates, saccharides such as molasses and blackstrap molasses, or their derivatives such as fatty acid esters, natural carbohydrates such as wheat, malt extract, rice, alcohols such as glycerol, mannitol, methanol, and ethanol, glucones, and pyruvic acid. , fatty acids such as acetic acid and citric acid, hydrocarbons such as normal paraffin and kerosene, and amino acids such as glycine, glutamic acid, glutamine, alanine, and asparagine, taking into account the assimilation ability of the microorganisms used. One or more of them may be appropriately selected and used. Nitrogen sources include meat extract, peptone, yeast extract, dried yeast, soybean hydrolyzate, soybean flour, milk casein,
Organic nitrogen compounds such as casamino acids, various amino acids, corn staple liquor, cotton seed meal or its hydrolysates, fruit meal or its hydrolysates, hydrolysates of other animals, plants, and microorganisms, ammonia, ammonium nitrate,
Select one or more of ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium acetate, nitrates such as sodium nitrate, and inorganic nitrogen compounds such as urea, taking into consideration the assimilation ability of the microorganisms used. and use it. Furthermore, as inorganic salts, magnesium, manganese, iron, zinc, copper, sodium,
phosphates and hydrochlorides such as calcium and potassium;
One or more types of nitrates, carbonates, nitrates, acetates, etc. are added as appropriate, and vegetable oil,
Antifoaming agents such as surfactants, trace amounts of growth promoting substances such as vitamins B1 , B2 , nicotinic acid, pantothenic acid, biotin and p-aminobenzoic acid may be added. Furthermore, when using microorganisms that exhibit nutritional requirements, it is necessary to add to the medium a substance that satisfies the growth of the microorganisms. The culture method is not particularly limited, but a culture method suitable for the microorganism used is usually selected from the usual culture methods such as shaking culture, aerated agitation culture, static culture, and continuous culture in a liquid medium containing the above-mentioned medium components. I will do it. Culture conditions may be selected appropriately depending on the type of microorganism and medium used, but when the purpose is to obtain bacterial cells, the pH at the start of culture is usually adjusted to about 6 to 8, and the culture is kept at a temperature of about 25 to 35℃. Culture is carried out under temperature conditions. The culture period may be a period sufficient for the growth of the microorganism used, and is usually 1 to 3 days. In addition, when culturing a microorganism to obtain the above-mentioned bacterial cells, a triazole compound and a ribose donor may be present in the medium to allow the microorganism to act on these reaction substrates. Enzyme source When carrying out the method of the present invention, the above-mentioned bacterial cells etc. are used as an enzyme source, and the reaction is carried out by bringing this into contact with a reaction substrate solution. shows. That is, microorganisms are cultured as described above, and the resulting culture is subjected to centrifugation, sedimentation, flocculation, and filtration (including pressure filtration, vacuum filtration, etc.).
Live microbial cells collected by a conventional method such as the above, or a treated microbial cell product obtained by subjecting viable microbial cells to appropriate treatments can be used as the enzyme source in the present invention. Here, the term "culture" refers to a state in which the culture medium and cultured bacterial cells are unseparated after cultivation. In addition, the bacterial cell treatment product is
Dried bacterial cells, cell membrane/wall-denatured bacterial cells, crushed bacterial cells, immobilized bacterial cells, bacterial cell extracts, protein fractions of bacterial cell extracts having enzyme activity involved in the production of ribavirin, which is the object of the present invention; It refers to its purified product, protein fraction, or immobilized product of its purified product. The method for obtaining the treated bacterial cell product is illustrated below. That is, physical treatments such as freeze-thaw treatment, freeze-drying treatment, ventilation drying treatment, acetone drying treatment, heating treatment under acidic or alkaline conditions, grinding treatment, ultrasonic treatment, osmotic pressure difference treatment, etc. means or enzyme treatments such as lysozyme, cell wall lytic enzymes, toluene,
By subjecting the bacterial extract to chemical or biochemical treatments such as contact treatment with a solvent such as xylene or butyl alcohol (butanol) or a surfactant, alone or in combination, for example, salting out treatment, By applying enzyme separation and purification methods such as isoelectric focusing treatment, organic solvent precipitation treatment, various chromatographic treatments, and dialysis treatment alone or in combination, live bacterial cells, bacterial cell extracts, or purified products thereof can be subjected to comprehensive treatment. A bacterial cell-treated product can be obtained by performing enzyme immobilization methods such as crosslinking treatment, adsorption treatment to a carrier, alone or in appropriate combinations. Reaction substrate The reaction substrate in the enzyme reaction of the present invention is 1, 2,
4-triazole-3-carboxamide and ribose donor. 1,2,4-triazole-3-carboxamide can be used in either a free form, an alkali salt such as a sodium salt, or an acid addition salt. As the ribose donor, any ribose derivative such as ribonucleoside, D-ribose, or various phosphoric acid esters thereof can be used. Furthermore, sugars other than such ribose derivatives that can be metabolized by the microorganism used and converted into ribose derivatives are regarded as ribose donors in the present invention. It should be noted that these ribose donors do not necessarily need to be added to the reaction substrate solution, and those contained in the bacterial cells etc. as their original components may be used. Furthermore, if such ribose donor as a bacterial cell component is consumed and reduced by the enzymatic reaction, it may be added from outside the system. Suitable ribose donors added from outside the system are illustrated below. That is, the base portion of the ribonucleoside may be any purine-based or pyrimidine-based base, and can be used regardless of whether it is derived from a natural product or chemically synthesized. Furthermore, even if the sugar hydroxyl group of ribonucleoside or D-ribose is unsubstituted or
one position, either the 3rd or 5th position hydroxyl group,
Two or all of them may be phosphoric esters such as monophosphoric esters, diphosphoric esters, or triphosphoric esters. Also,
These phosphoric acid esters may be in free form or may be common alkali salts such as sodium, potassium, calcium, magnesium, ammonium, triethylammonium and the like.
Specific examples of ribose donors include inosine, adenosine, guanosine, xanthosine, uridine,
Cytidine, 5'-inosinic acid, 5'-adenylic acid,
5'-guanylic acid, 5'-xanthylic acid, 5'-uridylic acid, 5'-cytidylic acid, 2'(3')-inosinic acid,
2′(3′)-adenylic acid, 2′(3′)-guanylic acid, 2
′(3′)
-xanthylic acid, 2'(3')-uridylic acid, 2'(3')
−
Cytidylic acid, D-ribose, D-ribose-1-
Examples include phosphoric acid. Examples of sugars that can be metabolized into ribose derivatives include glucose and sucrose. Reaction Substrate Solution The substrate solution used in the enzyme reaction of the present invention is basically an aqueous liquid in which the above-mentioned reaction substrate is dissolved or suspended in an aqueous medium. The aqueous liquid contains at least one or more of the above-mentioned triazole compounds and one or more of the above-mentioned ribose donors, and in addition to these reaction substrates, a phosphate ion donor system, an organic solvent, and an interface. Substances that promote enzyme reactions such as activators, metal salts, coenzymes, acids, bases, and sugars, substances that inhibit interfering enzyme activity, substances that improve the solubility of reaction substrates, and improve contact between enzymes and reaction substrates. It may also contain substances. Furthermore, the medium may contain the above-mentioned medium components that can be assimilated by the microorganisms used. As the aqueous medium, water or various buffers suitable for enzyme reactions (phosphate buffer, imidazole-hydrochloric acid buffer, veronal-hydrochloric acid buffer, Tris-hydrochloric acid buffer, etc.) can be used. As the phosphate ion donor system, any substance that can be dissociated into phosphate ions in an aqueous medium may be used, such as free phosphoric acid itself, inorganic phosphates, alkali metals such as sodium and potassium, calcium, Salts with alkaline earth metals such as magnesium and ammonium are preferably used. Phosphate ion donating systems include systems that can liberate phosphate ions in substrate solutions for enzyme reactions, such as combinations of various phosphate ester derivatives and phosphatases, combinations of nucleotides and nucleotidases, nucleobases, and ribose-1.
- A combination of phosphoric acid and phosphorylase can be used. The above-mentioned phosphoric acid donor system may be added from outside the system during the enzyme reaction, or may be contained as a component of the microorganism used. That is, as long as it is in a form that can be used for enzymatic reactions, the above-mentioned substances alone or in combination of two or more, or microorganisms containing the above-mentioned substances or their processed products can be used according to the present invention. These substances may be added separately to the reaction solution during the enzyme reaction, or these substances contained as bacterial cell components of the microorganism used may be used as they are. Examples of organic solvents include methanol, ethanol, propanol, butanol, pentanol, acetone, methyl ethyl ketone, ethyl acetate, toluene, tetrahydrofuran, dioxane, dimethyl sulfoxide, dimethyl acetamide, dimethyl formamide, 2-methoxyethanol, 2-ethoxy ethanol, 1 , 2-dimethoxyethane and the like. Contact Method The reaction of the present invention is achieved by bringing the enzyme source and reaction substrate into contact in an aqueous medium. The contact method may be selected as appropriate depending on the form of the enzyme source, but it is usually a batch method in which the enzyme source is suspended or dissolved in a reaction substrate solution and preferably stirred or shaken while heating, or a contact method is used. Mix with appropriate carriers, auxiliaries, and adsorbents as necessary,
Alternatively, a column method may be applied in which the reaction substrate solution is passed through the reaction substrate by supporting the reaction material in a column. In the case of the batch method, after the reaction, bacterial cells are collected by a conventional method such as filtration (including pressure filtration, vacuum filtration, etc.), centrifugation, sedimentation separation, flocculation separation, etc., and the reaction substrate is collected. It can be used repeatedly by contacting it with a solution. In the case of the column method, separation of bacterial cells and the like is not required, but it can be used repeatedly or continuously in the same way for enzymatic reactions. In addition, when culturing the microorganisms used,
A method in which a reaction substrate is added to the medium and the enzyme source and reaction substrate are reacted can also be adopted in the present invention. Concentrations or Added Amounts of Reaction Substrates and Enzyme Sources During the reaction, the substrate concentration of the reaction solution is not particularly limited, and a substrate concentration below the saturation concentration of each substrate in the aqueous medium used at the reaction temperature is usually adopted. The substrate concentration can also be increased by adding the above-mentioned organic solvent, surfactant, etc. to the reaction substrate solution. Alternatively, each substrate can be present in a suspended state at a saturation concentration or higher in the reaction solution, and each substrate can be dissolved as the reaction progresses. Alternatively, each substrate can be added sequentially during the reaction and maintained at an appropriate concentration. When each substrate is added and dissolved, the substrate concentration is 1,2,4-triazole-3-
For carboxamides or their salts, usually 5~
It is about 200mM, preferably about 10-100mM, and usually 0-300mM for ribose donors.
degree, preferably about 1 to 150 mM. The amount of the enzyme source to be used can be easily determined by a person skilled in the art through preliminary experiments, etc., taking into account the type of microorganism, its mode of use, reaction efficiency, economics, etc.; For (wet) bacterial cells, it is sufficient to use a substrate solution of 10 to 150 mg/ml, for dry bacterial cells, it is sufficient to use a substrate solution of 2 to 30 mg/ml, and for column methods, set the appropriate amount according to the batch method. can do. Reaction conditions The conditions for the reaction of the present invention are particularly limited, regardless of whether the microorganism used is under non-proliferation conditions or under growth conditions, as long as the reaction substrate reacts with the action of bacterial cells and ribavirin is efficiently produced. Not done. However, the reaction under non-growth conditions of the microorganism used is particularly efficient. As a method for conducting the reaction under conditions where microorganisms do not grow, the enzyme reaction temperature is set within a temperature range in which the microorganisms used do not grow (however, the temperature range does not inactivate the enzymes involved in the reaction of the present invention). ,
A method of subjecting the microorganisms to a state in which they cannot grow by physically, chemically or biochemically treating them as described above, and then subjecting them to a reaction; The method of adding a substance that inhibits the reaction to the reaction substrate solution may be used alone or in combination, but the method of manipulating the reaction temperature is particularly effective and simple. The reaction of the present invention proceeds in a temperature range of 28 to 80°C, but in consideration of practicality, a temperature range of 37 to 70°C is preferred, and a temperature of 45 to 60°C is particularly optimal. Note that at a temperature of 37° C. or higher, the reaction in which ribavirin produced by the reaction is decomposed by the action of bacterial cells or the like present in the reaction solution is suppressed, so the production efficiency of ribavirin is good. The liquid properties of the reaction substrate solution should be generally maintained within the range of PH4 to 10, preferably PH6 to 8.
When the PH fluctuates, it may be corrected to a preferred PH range using an acid such as hydrochloric acid, sulfuric acid, or phosphoric acid, or an alkali such as sodium hydroxide, potassium hydroxide, aqueous ammonia, or ammonia gas. The reaction time can be determined while checking the conversion rate of the reaction substrate to the target product, but in the case of a batch method, the reaction time is usually about 2 to 45 hours, preferably 24 to 36 hours, and in the case of a column method, the reaction time is about 2 to 45 hours. The reaction may be carried out by setting appropriate conditions according to the method. Separation and purification After the reaction, if necessary, isolate and remove bacterial cells using conventional methods such as filtration, centrifugation, or aggregation.
Subjected to ribavirin separation and purification process. Separation and purification of ribavirin may be carried out using known methods or applications thereof, such as various chromatography methods such as ion conversion chromatography, adsorption chromatography, partition chromatography, and gel filtration methods, countercurrent distribution, General separation and purification methods such as methods that utilize distribution between two liquid phases such as countercurrent extraction, and methods that utilize differences in solubility such as concentration, cooling, and addition of organic solvents may be used alone or in appropriate combinations. . Analysis In the examples of the present invention, ribavirin and 1,
Analysis of 2,4-triazole-3-carboxamide was performed by high performance liquid chromatography. When analyzed using the equipment and conditions shown below, ribavirin showed a retention time of 1,2,4-
Triazole-3-carboxamide retention time
It is eluted around 2.65 minutes, and the amount of each can be calculated from the calibration curve. Equipment: Shimadzu high-performance liquid chromatograph LC-3A model (manufactured by Shimadzu Corporation) Column: Micro Bondapak (μBONDAPAK) C 18 , 4.6 mm x 250 mm (manufactured by Nippon Waters Limited) Eluent: Contains 2% acetonitrile 20mM Tris monohydrochloric acid buffer (PH7.5) Flow rate: 1ml/min Measurement wavelength: 225nm Column operating temperature: room temperature [ ] Examples The present invention will be explained in more detail with reference to Examples, but these are This also represents one embodiment of the invention, and does not limit the scope of the present invention. Example 1 Each strain shown in Table 1 was inoculated into 50 ml of a 2% aqueous solution of powdered bouillon (manufactured by Kyokuto Pharmaceutical Industries, Ltd.) and incubated at 28°C for 24 hours.
After shaking culture for a period of time, the bacteria were collected by centrifugation, and sterilized water was added to obtain 5 ml of each bacterial cell suspension. 20mM 1,2,4-triazole-3-carboxamide, 20mM inosine and 25mM phosphate-
5 ml of each of the above bacterial cell suspensions were added to each 5 ml of an aqueous solution containing potassium (PH7.0), and the mixture was reacted at 60°C for 20 hours.
After the reaction was completed, the bacterial cells were removed by centrifugation, and the ribavirin production rate was analyzed, and the results were as shown in Table 1. Furthermore, the ribavirin production rate is 1,
It refers to the conversion rate (%) of 2,4-triazole-3-carboxamide to ribavirin.
【表】【table】
【表】
実施例 ２
シユウドモナス・プトレフアシエンス
ATCC8072を実施例１と同様に培養し（ただし、
培養液は各10mlとした。）、培養後、遠心分離によ
つて集菌し、各１mlの殺菌水を加えて菌体懸濁液
を得た。
この菌体懸濁液に20ｍＭ、１，２，４−トリア
ゾール−３−カルボキサミド、20ｍＭの第２表に
示す各種リボース供与体および25ｍＭりん酸一カ
リウムを含む水溶液（PH7.0）各１mlを添加し、
60℃、20時間反応した。反応終了後、遠心分離に
よつて除菌し、上澄液を分析したところリバビリ
ンの生成率は第２表に示すとおりであつた。[Table] Example 2 Pseudomonas putrefaciens
ATCC8072 was cultured in the same manner as in Example 1 (however,
The culture solution was 10 ml each. ), after culturing, the bacteria were collected by centrifugation, and 1 ml of sterilized water was added to each to obtain a bacterial cell suspension. To this cell suspension, 1 ml each of an aqueous solution (PH7.0) containing 20 mM 1,2,4-triazole-3-carboxamide, 20 mM of various ribose donors listed in Table 2, and 25 mM monopotassium phosphate was added. death,
The reaction was carried out at 60°C for 20 hours. After the reaction was completed, bacteria were removed by centrifugation, and the supernatant was analyzed, and the production rate of ribavirin was as shown in Table 2.
【表】【table】
【表】
実施例 ３
スタフイロコツカス・アウレウスIAM1011お
よびフラボバクテリウム・アルボレセンス
IFO3750を使用し、リボース供与体として第３表
に示すヌクレオチドを使用したほかは実施例２と
同一の方法で反応を行い、第３表に示す結果を得
た。[Table] Example 3 Staphylococcus aureus IAM1011 and Flavobacterium arborescens
The reaction was carried out in the same manner as in Example 2, except that IFO3750 was used and the nucleotide shown in Table 3 was used as the ribose donor, and the results shown in Table 3 were obtained.
【表】
実施例 ４
フラボバクテリウム・ルテセンスIFO3084を粉
末ブイヨン２％水溶液２に植菌し、28℃、22時
間振盪培養した。
培養終了後、遠心分離によつて集菌し、洗滌
後、殺菌水を加えて200mlの菌体懸濁液を得た。
50ｍM1，２，４−トリアゾール−３−カルボ
キサミド、75ｍＭイノシンおよび50ｍＭりん酸一
カリウムを含む水溶液800mlに前記菌体懸濁液を
加え、60℃、24時間反応したところ、リバビリン
生成率は、72.55％であつた。
反応液を遠心分離し菌体を除去した後、250ml
に減圧濃縮し、冷却した。生成したヒポキサンチ
ンおよびイノシンを除去後、カオチン交換樹脂
（H+型）を通過させ、この通過水洗液を活性炭に
吸着させた。活性炭カラムよりエタノール・アン
モニア溶液でリバビリンを溶出し、溶出後のエタ
ノールを除去した後、アニオン交換樹脂を通過さ
せ、通過水洗液を減圧濃縮して25mlとし、冷却し
た。冷却後、析出した結晶を分離・乾燥しリバビ
リンの結晶6.6ｇを得た。
実施例 ５
第４表に示す各菌株を粉末ブイヨン２％水溶液
各100mlに植菌し、30℃、22時間振盪培養後、集
菌し、それぞれに水を加えて菌体懸濁液各10mlを
得た。
20ｍM1，２，４−トリアゾール−３−カルボ
キサミド、30ｍM5′−ウリジル酸ナトリウムおよ
び25ｍＭりん酸−カリウムを含む水溶液（PH7.0）
各10mlに前記の菌体懸濁液を加え、45℃、23時間
反応した。反応終了後、遠心分離によつて菌体を
除去し、リバビリン生成率を分析したところ第４
表に示すとおりであつた。[Table] Example 4 Flavobacterium lutecens IFO3084 was inoculated into 2% aqueous powder broth solution 2, and cultured with shaking at 28°C for 22 hours. After culturing, the cells were collected by centrifugation, washed, and sterilized water was added to obtain 200 ml of cell suspension. When the above cell suspension was added to 800 ml of an aqueous solution containing 50 mM 1,2,4-triazole-3-carboxamide, 75 mM inosine, and 50 mM monopotassium phosphate and reacted at 60°C for 24 hours, the ribavirin production rate was 72.55%. It was hot. After centrifuging the reaction solution to remove bacterial cells, 250ml
The mixture was concentrated under reduced pressure and cooled. After removing the generated hypoxanthine and inosine, it was passed through a cation exchange resin (H + type), and the passed water washing solution was adsorbed on activated carbon. Ribavirin was eluted from the activated carbon column with an ethanol/ammonia solution, and after removing the eluted ethanol, it was passed through an anion exchange resin, and the washed water solution was concentrated under reduced pressure to 25 ml and cooled. After cooling, the precipitated crystals were separated and dried to obtain 6.6 g of ribavirin crystals. Example 5 Each strain shown in Table 4 was inoculated into 100 ml of a 2% aqueous powdered bouillon solution, cultured with shaking at 30°C for 22 hours, collected, and water was added to each to prepare 10 ml of a bacterial cell suspension. Obtained. Aqueous solution containing 20mM 1,2,4-triazole-3-carboxamide, 30mM sodium 5'-uridylate and 25mM potassium phosphate (PH7.0)
The above bacterial cell suspension was added to each 10ml and reacted at 45°C for 23 hours. After the reaction, the bacterial cells were removed by centrifugation and the ribavirin production rate was analyzed.
It was as shown in the table.
【表】
実施例 ６
第５表に示す各菌株を粉末ブイヨン（極東製薬
工業(株)製）２％水溶液各50mlに植菌し、28℃、24
時間振盪培養後、遠心分離によつて集菌し、殺菌
水を加えて菌体懸濁液各５mlを得た。
20ｍM1，２，４−トリアゾール−３−カルボ
キサミドおよび25ｍＭりん酸一カリウムを含む水
溶液（PH7.0）各５mlに前記菌体懸濁液各５mlを
加え45℃、24時間反応した。反応終了後、リバビ
リン生成率を分析したところ第５表に示すとおり
であつた。[Table] Example 6 Each strain shown in Table 5 was inoculated into 50 ml of a 2% aqueous solution of powdered bouillon (manufactured by Kyokuto Pharmaceutical Industries Co., Ltd.) and incubated at 28°C for 24 hours.
After shaking culture for a period of time, the bacteria were collected by centrifugation, and sterilized water was added to obtain 5 ml of each bacterial cell suspension. 5 ml each of the above cell suspension was added to 5 ml each of an aqueous solution (PH 7.0) containing 20 mM 1,2,4-triazole-3-carboxamide and 25 mM monopotassium phosphate, and reacted at 45°C for 24 hours. After the reaction was completed, the ribavirin production rate was analyzed and found to be as shown in Table 5.
Claims (1)
フラボバクテリウム属、エシエリヒア属、セルロ
モナス属、エンテロバクター属、ビブリオ属、エ
ルヴイニア属またはクレブシエラ属に属し、１，
２，４−トリアゾール−３−カルボキサミドとリ
ボース供与体であるヌクレオチドとからリバビリ
ンを生成する能力を有する微生物の作用により、
１，２，４−トリアゾール−３−カルボキサミド
またはその塩およびヌクレオチドまたはその塩を
水性媒体中で反応させてリバビリンを生成させ、
これを取得することを特徴とするリバビリンの製
造法。1 Pseudomonas spp., Staphylocotcus spp.
Belongs to the genus Flavobacterium, Escherichia, Cellulomonas, Enterobacter, Vibrio, Erwinia or Klebsiella, 1,
Through the action of microorganisms that have the ability to produce ribavirin from 2,4-triazole-3-carboxamide and nucleotides that are ribose donors,
reacting 1,2,4-triazole-3-carboxamide or a salt thereof and a nucleotide or a salt thereof in an aqueous medium to produce ribavirin;
A method for producing ribavirin, which is characterized by obtaining this.
Priority Applications (12)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10083282A JPS58216696A (en) | 1982-06-11 | 1982-06-11 | Preparation of ribavirin |
| US06/489,409 US4614719A (en) | 1982-04-30 | 1983-04-26 | Process for producing ribavirin |
| DE8383104130T DE3368641D1 (en) | 1982-04-30 | 1983-04-27 | Process for producing ribavirin |
| AT83104130T ATE24518T1 (en) | 1982-04-30 | 1983-04-27 | PROCESS FOR THE MANUFACTURE OF RIBAVIRIN. |
| EP83104130A EP0093401B1 (en) | 1982-04-30 | 1983-04-27 | Process for producing ribavirin |
| IL68516A IL68516A (en) | 1982-04-30 | 1983-04-28 | Process for producing ribavirin |
| HU831484A HU191292B (en) | 1982-04-30 | 1983-04-29 | Process for the production of ribavirine |
| ES83521984A ES8406090A1 (en) | 1982-04-30 | 1983-04-29 | Process for producing ribavirin. |
| IE992/83A IE54924B1 (en) | 1982-04-30 | 1983-04-29 | Process for producing ribavirin |
| CA000426998A CA1196591A (en) | 1982-04-30 | 1983-04-29 | Process for producing ribavirin |
| AR292883A AR231308A1 (en) | 1982-04-30 | 1983-04-29 | A PROCEDURE FOR PREPARING 1-BE-D-RIBOFURANOSIL-1,2,4-TRIAZOLCARBOXAMIDE RIVAVIRINE |
| KR1019830001834A KR870002167B1 (en) | 1982-04-30 | 1983-04-29 | Process for preparing ribavirin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10083282A JPS58216696A (en) | 1982-06-11 | 1982-06-11 | Preparation of ribavirin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS58216696A JPS58216696A (en) | 1983-12-16 |
| JPH0337919B2 true JPH0337919B2 (en) | 1991-06-07 |
Family
ID=14284286
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP10083282A Granted JPS58216696A (en) | 1982-04-30 | 1982-06-11 | Preparation of ribavirin |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS58216696A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57146593A (en) * | 1981-03-09 | 1982-09-10 | Ajinomoto Co Inc | Preparation of ribofuranosyltriazole derivative |
-
1982
- 1982-06-11 JP JP10083282A patent/JPS58216696A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57146593A (en) * | 1981-03-09 | 1982-09-10 | Ajinomoto Co Inc | Preparation of ribofuranosyltriazole derivative |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS58216696A (en) | 1983-12-16 |
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