JPH0314439B2 - - Google Patents
Info
- Publication number
- JPH0314439B2 JPH0314439B2 JP11738582A JP11738582A JPH0314439B2 JP H0314439 B2 JPH0314439 B2 JP H0314439B2 JP 11738582 A JP11738582 A JP 11738582A JP 11738582 A JP11738582 A JP 11738582A JP H0314439 B2 JPH0314439 B2 JP H0314439B2
- Authority
- JP
- Japan
- Prior art keywords
- reaction
- microorganisms
- ribavirin
- culture
- present
- 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
Links
- 244000005700 microbiome Species 0.000 claims description 77
- 238000006243 chemical reaction Methods 0.000 claims description 76
- 238000000034 method Methods 0.000 claims description 54
- 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 45
- 229960000329 ribavirin Drugs 0.000 claims description 44
- 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 44
- 230000001580 bacterial effect Effects 0.000 claims description 38
- 239000000126 substance Substances 0.000 claims description 21
- 238000004519 manufacturing process Methods 0.000 claims description 16
- 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 13
- ZEWJFUNFEABPGL-UHFFFAOYSA-N 1,2,4-triazole-3-carboxamide Chemical compound NC(=O)C=1N=CNN=1 ZEWJFUNFEABPGL-UHFFFAOYSA-N 0.000 claims description 11
- 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 11
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims description 11
- 150000003839 salts Chemical class 0.000 claims description 8
- 241000192041 Micrococcus Species 0.000 claims description 7
- 239000012736 aqueous medium Substances 0.000 claims description 7
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 6
- 241000186146 Brevibacterium Species 0.000 claims description 6
- 241000186216 Corynebacterium Species 0.000 claims description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims 2
- 229910052760 oxygen Inorganic materials 0.000 claims 2
- 239000001301 oxygen Substances 0.000 claims 2
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- 102000004190 Enzymes Human genes 0.000 description 30
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- -1 triazole compound Chemical class 0.000 description 17
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 12
- 239000002609 medium Substances 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 8
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-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
- 238000012258 culturing Methods 0.000 description 6
- 235000015097 nutrients Nutrition 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 229910021529 ammonia Inorganic materials 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 239000000306 component Substances 0.000 description 5
- 235000019441 ethanol Nutrition 0.000 description 5
- 239000000284 extract 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
- 238000000746 purification Methods 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
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- 108090000623 proteins and genes Proteins 0.000 description 4
- 235000000346 sugar Nutrition 0.000 description 4
- 239000004094 surface-active agent Substances 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
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-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
- 229910019142 PO4 Inorganic materials 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 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
- 229920002472 Starch Polymers 0.000 description 3
- YXJDFQJKERBOBM-TXICZTDVSA-N alpha-D-ribose 1-phosphate Chemical compound OC[C@H]1O[C@H](OP(O)(O)=O)[C@H](O)[C@@H]1O YXJDFQJKERBOBM-TXICZTDVSA-N 0.000 description 3
- 239000007864 aqueous solution Substances 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
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 3
- 239000006227 byproduct 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
- 238000005516 engineering process Methods 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N hydrochloric acid Substances Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 235000013372 meat Nutrition 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 108010009099 nucleoside phosphorylase Proteins 0.000 description 3
- 235000021317 phosphate Nutrition 0.000 description 3
- 229940085991 phosphate ion Drugs 0.000 description 3
- 239000012264 purified product Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 150000008163 sugars Chemical class 0.000 description 3
- 238000012360 testing method Methods 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
- 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 2
- 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 2
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-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
- 241000131747 Exiguobacterium acetylicum Species 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
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- 239000005715 Fructose Substances 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
- 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 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 241000191948 Kocuria rosea Species 0.000 description 2
- 241000191953 Kocuria varians Species 0.000 description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 2
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- 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
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- 239000004254 Ammonium phosphate Substances 0.000 description 1
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- 241000186063 Arthrobacter Species 0.000 description 1
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- 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
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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
利用する酵素的な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
シール−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）
参照）。
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、微生物の培養による方法および酵素的な
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（溶融性）、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２〜３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（以下、この方法を「培
養による公知方法」ということもある；特公昭54
−17830号公報、日本農芸化学会誌、50(9)、423〜
430（1976）参照）。本発明とこの公知方法とは本
質的に相異する技術である。すなわち、本発明の
方法においては微生物を酵素源とみなし、微生物
が増殖しない条件下で反応基質をこれらの微生物
の培養物、菌体または菌体処理物（以下、これら
を総活的に「菌体等」と称することもある。）の
触媒作用によつて反応させてリバビリンを合成す
るのに対し、培養による公知方法においては微生
物の代謝系を利用する目的で、十分に増殖しうる
条件下で微生物を培養し、培地中に添加された前
駆体であるトリアゾール化合物を代謝させてリボ
シル化させる点である。したがつて、本発明の方
法においては、酵素反応に際して微生物菌体は休
止ないし死滅した状態で使用され、基質溶液へ加
えるべき必須成分は反応基質であるトリアゾール
化合物のみかあるいはこれらに加えて酵素反応に
必要な無機物、補酵素類だけで十分であり、反応
条件は微生物が増殖しない条件、たとえば高温度
条件が最適なのである。これに対して前記の培養
による公知方法においては、微生物は盛んに増殖
している状態で使用され、培地成分としてはトリ
アゾール化合物に加えて使用微生物が十分に生育
しうるような炭素源、窒素源、無機物、その他の
栄養源を含有していなければならず、培養条件は
微生物が十分に生育しうる条件、すなわち比較的
低温条件でなければならないのである。また、使
用微生物は、本発明の方法を採用する場合には天
然よりの分離菌、公共機関の保存菌など通常の微
生物から広く選択して使用できるが、前記の従来
技術の方法を採用する場合には、糖源からリボー
ス供与体を生成させる意味でのヌクレオシド生産
性を有することが必要であり、アデニン要求性、
６−メルカプトグアニン抵抗性など特殊な性質を
有する微生物を使用しなければリバビリンの生産
量を増加することはできない。
また、酵素的な製造法としてはトリアゾール化
合物とリボース−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リース−１−りん
酸を反応基質として添加することが示されてい
る。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。この公知方法は、リボース供与体
としてリボース−１−りん酸を反応基質溶液に添
加する点および酵素源として記載されている微生
物の種類において本発明の方法とは全く異なる方
法である。
〔〕 発明の概要
要 旨
本発明者らは、微生物の培養物、菌体または菌
体処理物を酵素源とし、微生物の非増殖条件下に
酵素反応によつてリバビリンが生成することを初
めて知見し、この知見に基づいて本発明を完成し
た。
本発明は、ブレビバクテリウム属、コリネバク
テリウム属、ミクロコツカス属またはバチルス属
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とからリバビリンを生
成する反応を触媒する酵素系を含有し、かつ反応
系ヘリボース供与体の添加を必要としない微生物
の培養物、菌体または菌体処理物と、１，２，４
−トリアゾール３−カルボキサミドまたはその塩
とを前記微生物の非増殖条件下において水性媒体
中で接触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。
効 果
本発明における酵素反応液は組成が単純なの
で、微生物の栄養培地に比べて調製が簡便であ
り、栄養培地のようにあらかじめ殺菌する必要も
ない。また、微生物の非増殖条件下、たえば高温
条件下で反応を行う場合には反応液の雑菌汚染が
ほとんどないだけでなく、リバビリンの分解反応
が抑制されるのでいつたん合成されたリバビリン
は酵素反応液中に多量に蓄積する。本発明の方法
は酵素反応なので反応時間が短かく、副生物の生
成もほとんどない。したがつて、菌体等と分離後
の反応液中には主に末反応の原料化合物とリバビ
リンしか存在せず、リバビリンの単離精製が容易
である。さらに、本発明が菌体等の非増殖条件に
おける酵素反応であることによる副次的効果とし
ては、使用微生物をあらかじめ多量に培養して菌
体等を適宜な方法で存在しておけば、必要なとき
に必要な量を本発明の酵素反応に供することがで
きるなどの利点がある。
ちなみに、培養による公知方法の欠点は次のと
おりである。すなわち公知方法においてはリバビ
リンの製造は、微生物の増殖中に栄養培地中で行
われるので、まず微生物を増殖させるために各種
の栄養源を含有する培地を調製しなければなら
ず、種菌を植菌する前にこれらの培地を殺菌しな
ければならないなど前処理が煩雑である。また、
リバビリンの蓄積を目的とする、微生物の増殖を
伴う培養は、通常20〜40℃の常温で行われるの
で、常に雑菌汚染への配慮が必要であるばかりで
はなく、このような条件下ではリバビリン分解活
性も存在しているので生成したリバビリンも分解
してしまい目的物の収量を低下させる原因となつ
ていた。公知方法は微生物の増殖を伴う方法であ
るため、培養を２〜８日間という長期間にわたつ
て行わなければならず、各種のヌクレオシド、リ
バビリンのりん酸化物、その他の代謝産物が副生
する。したがつて培養液からリバビリンを回収す
るためには、原料化合物だけでなく、各種の副生
物とも分離しなければならず単離精製が煩雑であ
る。さらに、微生物はリバビリンの製造の度に培
養しなければなららない。
〔〕 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、その培養
物、菌体または菌体処理物が、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
との反応を触媒し、リバビリンを生成する酵素系
を含有するものであり、具体的にはブレビバクテ
リウム（Brevibacterium；以下、「B.」と略すこ
ともある。）属、コリネバクテリウム
（Corynebacterium；以下、「C.」と略すこともあ
る。）属、ミクロコツカス（Micrococcus；以下、
「M.」と略すこともある。）属またはバチルス
（Bacillus；以下、「Bac.」と略すこともある。）
属に属し、前記の酵素活性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。
前記の酵素活性の強い代表的菌株の一つとし
て、兵庫県西宮市の甲子園球場の砂より分離され
たAT−６−７株を挙げることができる。この菌
株の菌学的性質を以下に記載する。
Ａ 形態
(1) 細胞の形態および大きさ：短桿状、0.8〜
1.0×1.0〜1.2μｍ
(2) 胞子の形成：なし
(3) グラム染色性：陽性
Ｂ 各種培地における生育状態
(1) 肉汁寒天平板培養（28℃、48時間）
集落の形状：円形（Circular）
集落表面の***：扁平状（Flat）、平滑
（Smooth）
大きさ：２〜４mm
色調：黄色ないし桃黄色
(2) 肉汁寒天斜面培養（28℃、48時間）
生育：良好
生育の形：疣状（Echinulate）
(3) 肉汁液体培養28℃、48時間）
生育：表面に菌環（Ring）を形成し、やや
沈渣（Sediment）を生じる。
(4) 肉汁ゼラチン穿刺培養（20℃、６日間）：
層状（Straitiform）に液化する。
(5) リトマスミルク培地（28℃、４日間）：わ
ずかに凝固し、ペプトン化も見られる。
Ｃ 生理的性質
(1) 硝酸塩の還元（28℃、５日間）：還元性な
し。
(2) 硫化水素の生成（28℃、５日間）：生成し
ない。
(3) 澱粉の加水分解：分解性あり。
(4) カタラーゼ：陽性
(5) インドールの生成：生成しない。
(6) ペプトンおよびアルギニンからのアンモニ
アの生成：陰性
(7) メチルレツドテスト：陰性
(8) Ｖ−Ｐテスト：陽性
(9) 酵素に対する態度：好気的
(10) Ｏ−Ｆテスト（Hugh Leifson法による）：
Ｆ型（Fermention）
(11) 糖類からの酸の生成
陽性：グルコース、マンノース、フラクトー
ス、マルトース、サツカロース、トレハロ
ース
陰性：アラビノース、キシロース、ガラクト
ース、ソルビツト、イノシツト、グリセリ
ン
(12) 生育PH範囲：PH6.0〜9.0
(13) 生育最適温度：25〜37℃
以上の菌学的性質を、バージエーズ・マニユア
ル・オブ・デイタミネーテイブ・バクテリオロジ
ー（Bergey′s Manual of Determinative
Bacteriology）第７版（1957年）の分類基準に
より検索した。その結果、AT−６−７株はほと
んど球菌に近い短桿菌で、グラム陽性であり、フ
イラメントを形成せず、炭水化物より酸を生成す
ることによりブレビバクテリウム
（Breribacterium）属に属する菌株と同定し、ブ
レビバクテリウム・アセチリカム
（Brevibacterium acetylicum）AT−６−７と命
名した。
なお、以上の菌株の同定帰属はバージエーズ・
マニユアル・オブ・デイタミネーテイブ・バクテ
リオロジ−第７版によるものであり、分類基準の
変更などにより、異なる分類基準によつてこの菌
株の同定帰属が行われた場合には、他種あるいは
他属に属することもあり得るが、本発明において
上記のごとく命名された微生物は、少なくとも本
発明の目的とするリバビリンの製造に関与する酵
素を生産し、かつ前記の菌学的性質もしくはこれ
と均等の菌学的性質を基本的に有する微生物を包
含し、一義的に特定され得るものである。
この菌株について、昭和56年通商産業省告示第
178号に従つて工業技術院微生物工業技術研究所
に対して寄託申請を行い、昭和57年１月13日付け
で受託され、受託番号として微工研菌寄第6305号
（FERM Ｐ−6305）が付与されている。
また、上記菌株以外に本発明の目的とするリバ
ビリン製造のための酵素活性が強く、かつ当業者
が容易に入手できる菌株を以下に例示する。
ブレビバクテリウム・インペリアレ（B.
imperiale） ATCC 8365
コリネバクテリウム・エクイ（C.equi）
IAM 1038
バチルス・ズブチリス（Bac.subtilis）
ATCC 14593
バチルス・セレウス（Bac.cereus） IAM 1029
ミクロコツカス・ルテウス（M.luteus）
ATCC 4698
ミクロコツカス・ルテウス IAM 1056
ミクロコツカス・ヴアリアンス（M.varians）
IFO 3765
ATCC 399
ミクロコツカス・ロゼウス（M.roseus）
IFO 3768
ATCC 186
ミクロコツカス・カゼオリテイカス（M.
caseolyticus） IFO 3760
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およびIMA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である。
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の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ブレビバクテ
リウム属、コリネバクテリウム属、ミクロコツカ
ス属またはバチルス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
のような微生物の培養物、培養菌体またはその処
理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
本発明に使用する菌体等を調製するために、こ
れらの微生物を培養するに際しては、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、サツカロース、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、〓、米な
どの天然炭化水物、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、グルコン酸、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、無機塩として微量のマグネシ
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、硫
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。
培養は、前記培地成分を含有する液体培地中で
振盪培養、通気攪拌培養、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、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。
酵素源
以上のように微生物を培養した後、得られた培
養物、培養物から遠心分離、沈降分離、凝集分離
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塩のいずれ
も使用できる。
本発明においてリボース供与体とは、本発明の
使用微生物の菌体等の作用により、１，２，４−
トリアゾール−３−カルボキサミドにＤ−リボー
ス残基を転移しうるリボース誘導体であり、酵素
源として使用する菌体等にその菌体内成分として
概に含まれている物質であつて、本発明の反応に
関与する酵素の直接の反応基質のみならず本発明
の反応条件において上記反応基質に誘導されうる
物質をも包含する。このような物質としては各種
の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が挙げられる。
本発明は、前記のとおりリボース供与体を反応
系外より添加することなく、使用微生物の菌体内
成分として含まれるこれらの物質を利用する点に
一つの特徴を有するものである。
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
合物を添加し、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。
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
ん酸イオンに遊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
生物が菌体成分として含有し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
組み合せた系を、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
微生物が菌体成分として含有しているこれらの物
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。
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、反応液に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
上の基質を懸濁状態で存在させ、反応の進行に従
つて基質を溶解させることもできる。また、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。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、菌体等を非増殖条件
下、すなわち休止もしくは死滅菌体の状態で反応
に供すること以外は特に限定されない。
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。
本発明の反応において反応温度は上記のとおり
重要な条件であり、本発明を特徴づける要件の一
つである。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
しい。なお、最適の温度条件は他の反応条件によ
つて異るが、当業者であれば予備実験などによ
り、容易に決定することができる。
上記のような温度範囲で酵素反応を行うことに
より使用微生物の生育は大部分抑制される。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、たとえばイオン交換ク
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
−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１表に示すとおり
であつた。
[] BACKGROUND TECHNICAL FIELD OF THE INVENTION The present invention relates to an enzymatic 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, a broad-spectrum and potent antiviral against DNA and RNA viruses. It is known as a compound that exhibits action (Annals of the New York Academy of Sciences (Ann.
New York Acad. Sci.) 284 , 272-292 (1977)
reference). 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 property) 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 Application 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 culture for a long period of 2 to 3 days after the start of culture to produce and accumulate ribavirin in the medium (hereinafter, this method may also be referred to as ``known method using culture''). Yes; special public service in 1977
−17830 Publication, Journal of the Japanese Society of Agricultural Chemistry, 50 (9), 423~
430 (1976)). The present invention and this known method are essentially different technologies. In other words, in the method of the present invention, microorganisms are regarded as enzyme sources, and the reaction substrate is prepared from cultures, cells, or treated cells of these microorganisms (hereinafter, collectively referred to as "microorganisms") under conditions in which microorganisms do not proliferate. Ribavirin is synthesized by a reaction using the catalytic action of microorganisms (sometimes referred to as "microorganisms, etc."), whereas in the known culture-based method, the metabolic system of microorganisms is utilized, and ribavirin is synthesized under conditions that allow sufficient growth. The point is that microorganisms are cultured in a culture medium, and a triazole compound, which is a precursor added to the culture medium, is metabolized and ribosylated. Therefore, in the method of the present invention, the microbial cells are used in a dormant or dead state during the enzymatic reaction, and the essential component to be added to the substrate solution is only the triazole compound as the reaction substrate, or in addition to the triazole compound as the reaction substrate. The necessary inorganic substances and coenzymes are sufficient, and the optimal reaction conditions are conditions where microorganisms do not grow, such as high temperature conditions. On the other hand, in the known culture method described above, microorganisms are used in a state where they are actively proliferating, and the culture medium components include a carbon source and a nitrogen source that allow the microorganisms used to grow sufficiently, in addition to a triazole compound. The microorganisms must contain nutrients such as minerals, minerals, and other nutrients, and the culture conditions must be relatively low-temperature conditions that allow the microorganisms to grow sufficiently. In addition, when the method of the present invention is adopted, the microorganisms used can be widely selected from ordinary microorganisms such as naturally isolated bacteria and bacteria stored in public institutions, but when the method of the above-mentioned prior art is adopted For this purpose, it is necessary to have nucleoside productivity in the sense of generating ribose donors from sugar sources, and adenine requirement,
The production of ribavirin cannot be increased unless microorganisms with special properties such as resistance to 6-mercaptoguanine are used. Furthermore, as an enzymatic production method, a method is known in which a triazole compound and ribose-1-phosphate are reacted in the presence of nucleoside phosphorylase under conditions of pH 5 to 9 and temperature of 0 to 50°C (Unexamined Japanese Patent Publication No. (See Publication No. 50-29720). In this method, it has been shown that ribose-1-phosphate is added as a reaction substrate as a 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. This known method is completely different from the method of the present invention in that ribose-1-phosphate is added as a ribose donor to the reaction substrate solution and in the type of microorganism described as the enzyme source. [] Summary of the Invention The present inventors have discovered for the first time that ribavirin is produced by an enzymatic reaction under conditions in which microorganisms do not proliferate, using microorganism cultures, microbial cells, or processed microbial cells as enzyme sources. Based on this knowledge, the present invention was completed. The present invention relates to an enzyme system belonging to the genus Brevibacterium, Corynebacterium, Micrococcus or Bacillus that catalyzes the reaction of producing ribavirin from 1,2,4-triazole-3-carboxamide and a ribose donor. A microorganism culture, bacterial cells, or treated bacterial cells containing 1, 2, 4, and which does not require the addition of a helibose donor for the reaction system.
The present invention provides a method for producing ribavirin, which comprises producing ribavirin by contacting -triazole 3-carboxamide or a salt thereof in an aqueous medium under conditions in which the microorganisms do not grow. Effects Since the enzyme reaction solution of the present invention has a simple composition, it is easier to prepare than a nutrient medium for microorganisms, and there is no need to sterilize it in advance unlike a nutrient medium. Furthermore, when the reaction is carried out under conditions in which microorganisms do not proliferate, for example under high temperature conditions, not only is there almost no bacterial contamination of the reaction solution, but the decomposition reaction of ribavirin is inhibited, so the synthesized ribavirin is immediately A large amount accumulates in the reaction solution. Since the method of the present invention is an enzymatic reaction, the reaction time is short and almost no by-products are produced. Therefore, only the raw material compound of the final reaction and ribavirin are present in the reaction solution after separation from the bacterial cells, etc., and ribavirin can be easily isolated and purified. Furthermore, as a side effect of the present invention being an enzymatic reaction under non-proliferation conditions of bacterial cells, etc., if the microorganisms to be used are cultured in large quantities in advance and the bacterial cells etc. are present in an appropriate manner, the necessary It has the advantage that it can be used in the enzyme reaction of the present invention in the required amount at any time. Incidentally, the drawbacks of known methods using culture are as follows. In other words, in the known method, the production of ribavirin is carried out in a nutrient medium during the growth of microorganisms. Therefore, in order to grow the microorganisms, it is first necessary to prepare a medium containing various nutrient sources, and then inoculate the inoculum. Pretreatment is complicated, as these media must be sterilized before use. Also,
Cultivation involving the growth of microorganisms for the purpose of accumulating ribavirin is usually carried out at room temperature between 20 and 40°C, so not only must consideration be given to bacterial contamination, but also under these conditions ribavirin decomposes. Since it also has activity, the produced ribavirin is also degraded, causing a decrease in the yield of the target product. Since the known method involves the growth of microorganisms, the culture must be carried out for a long period of 2 to 8 days, and various nucleosides, ribavirin phosphates, and other metabolites are produced as by-products. Therefore, in order to recover ribavirin from the culture solution, it is necessary to separate not only the raw material compound but also various by-products, making isolation and purification complicated. Furthermore, microorganisms must be cultured each time ribavirin is manufactured. [] Specific description of the invention Microorganism used The microorganism used in the present invention is a microorganism whose culture, microbial cells, or processed microbial cells are capable of reacting with 1,2,4-triazole-3-carboxamide and a ribose donor. It contains an enzyme system that catalyzes and produces ribavirin, and specifically contains the genus Brevibacterium (hereinafter sometimes abbreviated as "B."), Corynebacterium (hereinafter, " (sometimes abbreviated as "C."), genus Micrococcus (hereinafter referred to as "Micrococcus").
Sometimes abbreviated as "M." ) genus or Bacillus (hereinafter sometimes abbreviated as "Bac.")
Examples include microorganisms belonging to the genus and having the above-mentioned enzyme activity. In the present invention, the type of strain used is not particularly limited as long as it has these basic properties. One of the representative strains with strong enzyme activity is the AT-6-7 strain, which was isolated from the sand of Koshien Stadium in Nishinomiya City, Hyogo Prefecture. The mycological properties of this strain are described below. A Morphology (1) Cell morphology and size: short rod-shaped, 0.8~
1.0×1.0-1.2μm (2) Spore formation: None (3) Gram staining: Positive B Growth status in various media (1) Broth agar plate culture (28℃, 48 hours) Colony shape: Circular Ridges on colony surface: Flat, smooth Size: 2 to 4 mm Color: Yellow to pinkish yellow (2) Juicy agar slant culture (28℃, 48 hours) Growth: Good Growth shape: Wart-like (Echinulate) (3) Meat juice liquid culture 28℃, 48 hours) Growth: Forms a bacterial ring on the surface and produces a slight sediment. (4) Meat juice gelatin puncture culture (20℃, 6 days):
Liquefies in Straitiform. (5) Litmus milk medium (28°C, 4 days): Slight coagulation and peptonization is also observed. C Physiological properties (1) Reduction of nitrate (28°C, 5 days): No reducibility. (2) Hydrogen sulfide generation (28℃, 5 days): Not generated. (3) Starch hydrolysis: Degradable. (4) Catalase: Positive (5) Indole production: Not produced. (6) Formation of ammonia from peptone and arginine: negative (7) Methylred test: negative (8) V-P test: positive (9) Attitude towards enzymes: aerobic (10) O-F test (Hugh (by Leifson method):
Type F (Fermention) (11) Production of acid from sugars Positive: glucose, mannose, fructose, maltose, sutucarose, trehalose Negative: arabinose, xylose, galactose, sorbitol, inosit, glycerin (12) Growth PH range: PH6.0 ~9.0 (13) Optimum growth temperature: 25-37℃
The search was conducted using the classification criteria of Bacteriology, 7th edition (1957). As a result, strain AT-6-7 was identified as a strain belonging to the Brevibacterium genus because it is a short bacillus, almost like a coccus, is Gram-positive, does not form filaments, and produces acid from carbohydrates. , and named Brevibacterium acetylicum AT-6-7. The identification and attribution of the above strains is from Virgies.
This is based on the 7th edition of the Manual of Determinative Bacteriology, and if the identification and attribution of this strain is made using different classification criteria due to changes in classification criteria, it may be different from other species or other species. Although they may belong to the genus, the microorganisms named above in the present invention produce at least an enzyme involved in the production of ribavirin, which is the object of the present invention, and have the above-mentioned mycological properties or equivalents thereof. It includes microorganisms that basically have the mycological properties of the following, and can be uniquely identified. Regarding this strain, the 1981 Ministry of International Trade and Industry Notification No.
In accordance with No. 178, we filed an application for deposit with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and the deposit was made on January 13, 1981, with the deposit number being FERM P-6305. has been granted. In addition to the above-mentioned strains, the following are examples of strains that have strong enzymatic activity for producing ribavirin, which is the object of the present invention, and that are easily available to those skilled in the art. Brevibacterium imperiale (B.
imperiale) ATCC 8365 Corynebacterium equi (C.equi)
IAM 1038 Bacillus subtilis
ATCC 14593 Bac.cereus IAM 1029 M.luteus
ATCC 4698 Micrococcus luteus IAM 1056 Micrococcus varians (M.varians)
IFO 3765 ATCC 399 Micrococcus roseus (M.roseus)
IFO 3768 ATCC 186 Micrococcus caseoliticus (M.
caseolyticus) IFO 3760 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.
These numbers indicate the deposit numbers at the University of Tokyo), and these strains are stored in the list of stored strains of the depositary institution. In addition, strains with IFO and IMA numbers are listed in the Japan FCC Catalog of Microorganisms (JFCC Catalog of Microorganisms).
It is a preserved bacterium listed in ``Cultures''. 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 strains thus derived can also be used in the present invention, as long as they do not lose the enzymatic activity involved in the production of ribavirin, which is the object of the present invention. Furthermore, the gene of the enzyme system involved in ribavirin production, which is the object of the present invention, obtained from the strain preferably used in the present invention as described above is a gene of the genus Brevibacterium, Corynebacterium, Micrococcus or Bacillus. If the microorganism is taken up by other microorganisms and such traits are expressed, the method of using the culture, cultured cells, or processed product of such microorganism for the purpose of the present invention is not included in the present invention. Ru. Culture When culturing these microorganisms in order to prepare microbial cells and the like used in the present invention, the medium and culture method used are not particularly limited as long as these microorganisms 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 sugars such as glucose, fructose, maltose, galactose, ribose, sutucarose, starch, starch hydrolysates, molasses, blackstrap molasses, or derivatives thereof such as fatty acid esters, wheat, glutinous rice, and rice. natural carbohydrates, alcohols such as glycerol, mannitol, methanol, and ethanol, fatty acids such as gluconic acid, pyruvic acid, acetic acid, and citric acid, hydrocarbons such as normal paraffin and kerosene, glycine, glutamic acid, glutamine, and alanine. , amino acids such as asparagine, etc., one or more types may be appropriately selected and used from among general carbon sources, taking into account the assimilation ability of the microorganisms used. Nitrogen sources include meat extract, peptone, yeast extract, dried yeast, soybean hydrolyzate, soybean flour, milk casein, casamino acids,
Various amino acids, cornstarch liquor, corn seed meal or its hydrolyzate, fruit meal or its hydrolyzate, other animals,
Organic nitrogen compounds such as hydrolysates of plants and microorganisms, ammonium salts such as ammonia, ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium acetate, nitrates such as sodium nitrate,
Considering the assimilation ability of the microorganisms used, one or more types are appropriately selected and used from inorganic nitrogen compounds such as urea. Furthermore, one or more of phosphates, hydrochlorides, sulfates, carbonates, nitrates, acetates, etc. of magnesium, manganese, iron, zinc, copper, sodium, calcium, potassium, etc., as appropriate, are added as inorganic salts. If necessary, antifoaming agents such as vegetable oil and surfactants, and small 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. Cultivation is carried out in a liquid medium containing the above-mentioned medium components by selecting a culture method suitable for the microorganism used from among conventional culture methods such as shaking culture, aerated agitation culture, static culture, and continuous culture. Culture conditions may be appropriately selected depending on the type of microorganism and medium used, but the pH at the start of culture is usually adjusted to about 6 to 8, and culture is carried out at a temperature of about 25 to 35°C. The culture period may be a period sufficient for the growth of the microorganism used, and is usually 1 to 3 days. Enzyme source After culturing microorganisms as described above, the obtained culture, viable bacterial cells collected from the culture by conventional methods such as centrifugation, sedimentation, and flocculation, or viable bacterial cells as appropriate. A treated bacterial cell product obtained by such treatment can be used as an 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 treated bacterial cell product can be obtained by applying enzyme immobilization means such as cross-linking treatment, adsorption treatment to a carrier, etc. 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 or a salt such as a sodium salt. In the present invention, the ribose donor refers to 1,2,4-
It is a ribose derivative capable of transferring a D-ribose residue to triazole-3-carboxamide, and is a substance generally contained as an intracellular component in bacterial cells used as an enzyme source, and is suitable for the reaction of the present invention. It includes not only direct reaction substrates of the enzymes involved, but also substances that can be induced into the reaction substrates under the reaction conditions of the present invention. Such substances include various ribonucleosides or D-ribose or various phosphoric acid esters thereof. One of the features of the present invention is that, as described above, the ribose donor is not added from outside the reaction system, but rather these substances contained as internal components of the microorganism used are utilized. 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. At least the above-mentioned triazole compound is added to the aqueous solution, and in addition to these reaction substrates, phosphate ion donor systems, organic solvents, surfactants, metal salts, coenzymes, acids, bases, sugars, etc. are used for enzymatic reactions. It may contain a substance that promotes the activity of the enzyme, a substance that inhibits the interfering enzyme activity, a substance that improves the solubility of the reaction substrate, a substance that improves the contact between the enzyme and the reaction substrate, etc. 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 liberated 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 donating system may be added from outside the system during the enzymatic reaction, or may be contained as a bacterial component in the microorganism used. That is, as long as it is in a form that can be used for the enzymatic reaction, the above-mentioned substances alone or a combination of two or more of them, or microorganisms containing the above-mentioned substances or their processed products can be used in the enzymatic reaction of the present invention. In this case, these substances may be added separately to the reaction solution, or the substances contained in the microorganism used as bacterial cell components 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. Concentrations or Added Amounts of Reaction Substrate and Enzyme Source During the reaction, there is no particular restriction on the substrate concentration in the reaction solution, and a substrate concentration below the saturation concentration of the substrate in the aqueous medium used at the reaction temperature is usually adopted. The substrate concentration can also be increased by adding the aforementioned organic solvents, surfactants, etc. to the substrate solution. Alternatively, the substrate may be present in a suspended state at a saturation concentration or higher in the reaction solution, and the substrate may be dissolved as the reaction progresses. Alternatively, the substrate can be added sequentially during the reaction and maintained at an appropriate concentration. When adding and dissolving the substrate, the substrate concentration is usually about 5 to 200 mM for 1,2,4-triazole-3-carboxamide or its salt;
Preferably it is about 10-100mM. 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 not particularly limited, except that the bacterial cells are subjected to the reaction under non-proliferation conditions, that is, in the state of resting or dead sterilized cells. 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. In the reaction of the present invention, the reaction temperature is an important condition as described above, and is one of the requirements characterizing the present invention. The reaction proceeds in the range of 28 to 80°C, but in consideration of practicality, the range of 37 to 70°C is preferable. Note that the optimal temperature conditions vary depending on other reaction conditions, but can be easily determined by those skilled in the art through preliminary experiments and the like. By carrying out the enzymatic reaction within the above temperature range, the growth of the microorganisms used is largely suppressed. 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 inhibited, 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. 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. Ribavirin can be separated and purified by known methods or by applying them, such as various chromatography techniques such as ion exchange chromatography, adsorption chromatography, partition chromatography, gel filtration, 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-hydrochloric 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 below. 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 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 1.
【表】
実施例 ２
1.5％酵母エキス培地（PH7.5）５にブレビバ
クテリウム・アセチリカムAT−６−７（微工研
菌寄第6305号）の前培養液250mlの植菌し、28℃、
24時間培養した。培養液より遠心分離によつて菌
体を得、500mlの菌体懸濁液とした。
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有す
る溶液（PH7.0）500mlに前記菌体懸濁液500mlを
加え、45℃、24時間反応した。この反応液のリバ
ビリン生成率は24.38％でであつた。
菌体除去液を陽イオン交換樹脂（水素型）処理
して、未反応の１，２，４−トリアゾール−３−
カルボキサミドを除去後、活性炭にリバビリン溶
液を吸着した。２％アンモニアを含む50％エチル
アルコール溶液でリバビリンを溶出後アルコール
を溜去し、陰イオン交換樹脂（塩基型）を通過さ
せた。通過液を濃縮し、リバビリンの結晶475mg
を得た。[Table] Example 2 250 ml of a preculture of Brevibacterium acetylicum AT-6-7 (Feikoken Bibori No. 6305) was inoculated into 1.5% yeast extract medium (PH7.5) and incubated at 28°C. ,
Cultured for 24 hours. Bacterial cells were obtained from the culture solution by centrifugation, and a 500 ml cell suspension was prepared. 500 ml of the above cell suspension was added to 500 ml of a solution (PH7.0) containing 20 mM 1,2,4-triazole-3-carboxamide and 25 mM monopotassium phosphate, and reacted at 45°C for 24 hours. The ribavirin production rate of this reaction solution was 24.38%. The bacterial cell removal solution is treated with a cation exchange resin (hydrogen type) to remove unreacted 1,2,4-triazole-3-
After removing the carboxamide, the ribavirin solution was adsorbed onto activated carbon. After eluting ribavirin with a 50% ethyl alcohol solution containing 2% ammonia, the alcohol was distilled off and the mixture was passed through an anion exchange resin (base type). Concentrate the permeate to obtain 475 mg of ribavirin crystals.
I got it.
Claims (1)
属、ミクロコツカス属またはバチルス属に属し、
１，２，４−トリアゾール−３−カルボキサミド
とリボース供与体とからリバビリンを生成する反
応を触媒する酸素系を含有し、かつ反応系ヘリボ
ース供与体の添加を必要としない微生物の培養
物、菌体または菌体処理物と、１，２，４−トリ
アゾール−３−カルボキサミドまたはその塩とを
前記微生物の非増殖条件下において水性媒体中で
接触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。 ２ 酸素反応を微生物の非増殖条件下において行
う方法が、反応液を微生物の増殖できない温度条
件に保持して反応を行う方法、微生物菌体をあら
かじめ該微生物が休止もしくは死滅する方法によ
つて処理し、これを用いて反応を行う方法、およ
び反応液に微生物の増殖を阻害する物質を添加し
て反応を行う方法からなる群より選ばれた一種の
方法または二種以上を組み合せた方法である特許
請求の範囲第１項記載のリバビリンの製造法。 ３ 微生物の非増殖条件が、37〜70℃の温度条件
である特許請求の範囲第１または２項記載のリバ
ビリンの製造法。[Scope of Claims] 1 Belongs to the genus Brevibacterium, the genus Corynebacterium, the genus Micrococcus or the genus Bacillus,
A culture or bacterial cell of a microorganism that contains an oxygen system that catalyzes the reaction of producing ribavirin from 1,2,4-triazole-3-carboxamide and a ribose donor, and that does not require the addition of a helibose donor to the reaction system. Alternatively, ribavirin is obtained by contacting the bacterial cell-treated product with 1,2,4-triazole-3-carboxamide or a salt thereof in an aqueous medium under conditions in which the microorganism does not grow. A method for producing ribavirin. 2. A method in which the oxygen reaction is carried out under conditions in which microorganisms do not grow, a method in which the reaction is carried out while the reaction solution is maintained at a temperature condition that does not allow the growth of microorganisms, and a method in which the microorganism cells are treated in advance by a method in which the microorganisms are suspended or killed. It is one type of method selected from the group consisting of a method of carrying out a reaction using this, and a method of carrying out a reaction by adding a substance that inhibits the growth of microorganisms to the reaction solution, or a method that combines two or more types. A method for producing ribavirin according to claim 1. 3. The method for producing ribavirin according to claim 1 or 2, wherein the microorganism non-growth condition is a temperature condition of 37 to 70°C.
Priority Applications (12)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11738582A JPS596895A (en) | 1982-07-05 | 1982-07-05 | 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 |
|---|---|---|---|
| JP11738582A JPS596895A (en) | 1982-07-05 | 1982-07-05 | Preparation of ribavirin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS596895A JPS596895A (en) | 1984-01-13 |
| JPH0314439B2 true JPH0314439B2 (en) | 1991-02-26 |
Family
ID=14710334
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11738582A Granted JPS596895A (en) | 1982-04-30 | 1982-07-05 | Preparation of ribavirin |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS596895A (en) |
-
1982
- 1982-07-05 JP JP11738582A patent/JPS596895A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS596895A (en) | 1984-01-13 |
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