JP3586924B2 - Method for producing 5-methyluridine - Google Patents
Method for producing 5-methyluridine Download PDFInfo
- Publication number
- JP3586924B2 JP3586924B2 JP9286195A JP9286195A JP3586924B2 JP 3586924 B2 JP3586924 B2 JP 3586924B2 JP 9286195 A JP9286195 A JP 9286195A JP 9286195 A JP9286195 A JP 9286195A JP 3586924 B2 JP3586924 B2 JP 3586924B2
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- Prior art keywords
- methyluridine
- crystals
- methyluracil
- microorganism
- crystal
- Prior art date
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- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 title claims description 86
- DWRXFEITVBNRMK-JXOAFFINSA-N ribothymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 DWRXFEITVBNRMK-JXOAFFINSA-N 0.000 title claims description 86
- 238000004519 manufacturing process Methods 0.000 title claims description 10
- 239000013078 crystal Substances 0.000 claims description 107
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims description 76
- 239000012535 impurity Substances 0.000 claims description 36
- 244000005700 microbiome Species 0.000 claims description 31
- 238000000034 method Methods 0.000 claims description 30
- 238000000926 separation method Methods 0.000 claims description 22
- 239000012533 medium component Substances 0.000 claims description 16
- 238000004062 sedimentation Methods 0.000 claims description 14
- 238000006911 enzymatic reaction Methods 0.000 claims description 12
- 150000003839 salts Chemical class 0.000 claims description 12
- 239000002777 nucleoside Substances 0.000 claims description 11
- 150000003833 nucleoside derivatives Chemical class 0.000 claims description 11
- 238000010908 decantation Methods 0.000 claims description 8
- 229910052816 inorganic phosphate Inorganic materials 0.000 claims description 8
- 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 claims description 5
- 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 2
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- -1 ribose monophosphate Chemical class 0.000 claims description 2
- 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 38
- 239000000243 solution Substances 0.000 description 32
- 102000039446 nucleic acids Human genes 0.000 description 21
- 108020004707 nucleic acids Proteins 0.000 description 21
- 150000007523 nucleic acids Chemical class 0.000 description 21
- 238000006243 chemical reaction Methods 0.000 description 20
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 19
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 19
- 229940029575 guanosine Drugs 0.000 description 19
- 229940113082 thymine Drugs 0.000 description 18
- 238000002425 crystallisation Methods 0.000 description 17
- 230000008025 crystallization Effects 0.000 description 17
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 13
- 239000012452 mother liquor Substances 0.000 description 12
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- 238000004113 cell culture Methods 0.000 description 10
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- 239000000758 substrate Substances 0.000 description 10
- 238000001914 filtration Methods 0.000 description 9
- 239000012530 fluid Substances 0.000 description 9
- 239000002994 raw material Substances 0.000 description 7
- 241000589565 Flavobacterium Species 0.000 description 6
- 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 6
- 239000012141 concentrate Substances 0.000 description 6
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 6
- 239000013081 microcrystal Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 5
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- 241000191938 Micrococcus luteus Species 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
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- 239000007788 liquid Substances 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 3
- 239000006227 byproduct Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 3
- 229940045145 uridine Drugs 0.000 description 3
- 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 2
- ODDDVFDZBGTKDX-VPCXQMTMSA-N 1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)-2-methyloxolan-2-yl]pyrimidine-2,4-dione Chemical compound C1=CC(=O)NC(=O)N1[C@]1(C)O[C@H](CO)[C@@H](O)[C@H]1O ODDDVFDZBGTKDX-VPCXQMTMSA-N 0.000 description 2
- AFBXRHFIVUFPES-UHFFFAOYSA-N 5-methyl-1h-pyrimidine-2,4-dione Chemical compound CC1=CNC(=O)NC1=O.CC1=CNC(=O)NC1=O AFBXRHFIVUFPES-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 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
- 230000001580 bacterial effect Effects 0.000 description 2
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- 239000002904 solvent Substances 0.000 description 2
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- 238000003786 synthesis reaction Methods 0.000 description 2
- 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
- 229940124321 AIDS medicine Drugs 0.000 description 1
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- 241000589291 Acinetobacter Species 0.000 description 1
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- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 241000186321 Cellulomonas Species 0.000 description 1
- 241000186220 Cellulomonas flavigena Species 0.000 description 1
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- 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 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- UBORTCNDUKBEOP-UHFFFAOYSA-N L-xanthosine Natural products OC1C(O)C(CO)OC1N1C(NC(=O)NC2=O)=C2N=C1 UBORTCNDUKBEOP-UHFFFAOYSA-N 0.000 description 1
- 241001467578 Microbacterium Species 0.000 description 1
- 241001467566 Microbacterium lacticum Species 0.000 description 1
- 241000192041 Micrococcus Species 0.000 description 1
- 241000203720 Pimelobacter simplex Species 0.000 description 1
- 241001162968 Sarsina Species 0.000 description 1
- UBORTCNDUKBEOP-HAVMAKPUSA-N Xanthosine Natural products O[C@@H]1[C@H](O)[C@H](CO)O[C@H]1N1C(NC(=O)NC2=O)=C2N=C1 UBORTCNDUKBEOP-HAVMAKPUSA-N 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
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- 229910001410 inorganic ion Inorganic materials 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 238000000634 powder X-ray diffraction Methods 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 230000003134 recirculating effect Effects 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
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- UBORTCNDUKBEOP-UUOKFMHZSA-N xanthosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(NC(=O)NC2=O)=C2N=C1 UBORTCNDUKBEOP-UUOKFMHZSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- HBOMLICNUCNMMY-XLPZGREQSA-N zidovudine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-XLPZGREQSA-N 0.000 description 1
- 229960002555 zidovudine Drugs 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
【０００１】
【産業上の利用分野】
本発明は、医薬品原料として有用な５−メチルウリジン（５ーｍｅｔｈｙｌｕｒｉｄｉｎｅ）の製造方法に関するものである。５−メチルウリジンは、エイズ薬として市販されているアジドチミジン、また同じく臨床試験中のｄ４Ｔ（２’，３’ーｄｉｄｅｈｙｄｒｏｘｙ−２’，３’−ｄｉｄｅｏｘｙｔｈｙｍｉｄｉｎｅ）の合成中間体として有望視されている。
【０００２】
【従来の技術】
５−メチルウリジンは従来、化学的合成法で製造されており、そこでの精製は、主として、メタノール、エタノール等の有機溶剤を用いた結晶化により行われていた。（Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．，７８，２１１７（１９５６）；特開昭６３ー６３６６８；Ｈｅｌｖｅｔｉｃａ Ｃｈｉｍｉｃａ Ａｃｔａ、２１７９（１９８０）；Ｓｙｎｔｈｅｓｉｓ，２５９（１９８２））
【０００３】
これらの技術は、核酸を原料とし、微生物を用いて変換する方法とは副生する不純物が異なるため、精製を含めた５−メチルウリジンの製造方法としては参考にならない。
【０００４】
微生物を利用する５−メチルウリジンの製造方法としては、ヌクレオシド、またはリボースー１ーリン酸に５ーメチルウラシルを微生物存在下に作用させる事により製造する方法が知られている。（特開平２ー２３８８２）
【０００５】
しかしながら、この特許には５−メチルウリジンの精製法に関する具体的な記述が無く、製品としての５−メチルウリジンを製造するには不十分である。
【０００６】
とりわけ、５−メチルウリジンの大きな結晶を晶析により得る事は、結晶の分離性を良くして分離に必要な装置の規模を小さくする事ができ、また、不純物との分離も良くなり、高純度の製品を得るには必要不可欠であるが、それに関するなんらの記述もされていない。
【０００７】
通常、結晶成長には過飽和の大きさと不純物が大きな影響を与え、とりわけ、不純物の組成は晶析せんとする系によって全く事なり、この制御が晶析の可不可を決める重要な鍵であり、与えられた系での特徴的な制御が必要である場合が多い。
【０００８】
よって、微生物を利用した５−メチルウリジンの場合、生物化学的生産に必要な情報は公知であるが、反応液よりの精製については、独自に方法を開発する必要があった。
【０００９】
反応液より、若干の前処理を経て濃縮により５−メチルウリジンを晶析した所、２０から３０マイクロメーター（μｍ）の大きさの結晶しか得られず、これでは分離速度が非常に遅く、分離装置が大きな物になる事が判明した。更に、この大きさでは、後述する結晶の粒度の差を利用した不純物の分離には全く不適切である事が判明した。
【００１０】
５−メチルウリジンは微生物を用い、核酸類を原料として、酵素反応により製造することができるが、そのとき反応生成物中には、未反応物である核酸類および副生物である核酸類が不純物として含まれている。５−メチルウリジンを製造するのに５ーメチルウラシル（チミン）とグアノシンを原料として用いる場合、反応液中の不純物としては、主としてチミン、グアニン、グアノシン、および、２−Ａｍｉｎｏ−７−β−Ｄ−ｒｉｂｏｆｕｒａｎｏｓｙｌ−７Ｈ−ｐｕｒｉｎｅ−６（１Ｈ）ｏｎｅ（シュドグアノシン）が存在する。このうち、例えば、チミンは温度、ｐＨ等に対する溶解度変化のパターンが５−メチルウリジンに類似しているため、結晶化によって除去する事は難しい。また、イオン解離パターンも類似しているのでイオン交換樹脂等の処理による除去も困難である。
【００１１】
これら核酸系の不純物は、核酸を原料とした微生物による製造法に特有のものであり、公知の知識を活用する事ができない。また、公知文献ではこれらを分離する方法は記述されていない。
【００１２】
【発明が解決しようとする課題】
５−メチルウリジンを効率よく生産し、かつ、純度の高い物になるよう、微生物を用いた反応液からの５−メチルウリジン結晶の粒度を大きくして結晶と母液の分離効率を高め、さらには、この結晶と他の不純物結晶との分離効率をよくする方法を開発する。
【００１３】
【課題を解決するための手段】
発明者は、微生物を培養し、核酸類を原料として酵素反応にて５−メチルウリジンを製造し、その晶析お呼び分離方法を検討した際、微生物の培養液の処理方法により、後工程の５−メチルウリジンの晶析で得られる結晶の粒度に大きな差のある事を見いだし、本発明を完成した。すなわち、本発明は、リボース−１−燐酸若しくはその塩及び５−メチルウラシルから５−メチルウリジンを生成する能力を有する微生物またはヌクレオシド、無機燐酸若しくはその塩及び５−メチルウラシルから５−メチルウリジンを生成する能力を有する微生物を用い、酵素反応にて５−メチルウリジンを製造する方法において、該微生物を培養した後、
（イ）該培地成分の一部、または全部を除去し、次いで
（ロ）該微生物にリボース−１−燐酸若しくはその塩及び５−メチルウラシルまたはヌクレオシド、無機燐酸若しくはその塩及び５−メチルウラシルを作用せしめ、５−メチルウリジンを生成させた後、次いで
（ハ）生成した５−メチルウリジンを晶析し、分離する
ことを特徴とする５−メチルウリジンの製造方法に関するものである。
【００１４】
本発明に用いられる微生物は、リボース−１−燐酸若しくはその塩及び５−メチルウラシルから５−メチルウリジンを生成する能力を有する微生物またはヌクレオシド、無機燐酸若しくはその塩及び５−メチルウラシルから５−メチルウリジンを生成する能力を有する微生物であれば、使用可能である。例えば、アクロモバクター属、アシネトバクター属、エアロモナス属等、特許公報特開平２−２３８８２号に記載の微生物を挙げることができる。特に好ましい微生物としては
、アースロバクター属、セルロモナス属、フラボバクテリウム属、クレブジェラ属、ミクロバクテリウム属、ミクロコッカス属、サルシナ属に属する微生物を例示することができる。その具体例として、下記の微生物が挙げられる。
アースロハ゛クター シンフ゜レックス(Arthrobacter simplex) FERM P-10068
セルロモナス フラヒ゛ケ゛ナ(Cellulomonas flavigena) ATCC 486
フラホ゛ハ゛クテリウム レナナム(Flavobacterium rhenanum) FERM BP-1862
クレフ゛シ゛ェラ ニューモニエ(Klebsiella pneumoniae) ATCC 8308
ミクロハ゛クテリウム ラクチカム(Microbacterium lacticum) ATCC 8180
ミクロコッカス ルテウス(Micrococcus luteus) FERM P-7399
サルシナ ルテア(Sarcina lutea) FERM P-7400
【００１５】
上記微生物の培養は、炭素源、窒素源、Ｐ、Ｓ、Ｆｅ、Ｍｎ等の無機イオン、さらに必要ならば、ビタミン等の微量栄養素または蛋白分解物、酵母エキスのような有機窒素源を含有する通常の培地を用い、通常の培養方法で行なえば良い。
【００１６】
得られた微生物の培養液からの培地成分の除去方法としては、培養液を自然沈降後の上澄み除去、遠心分離ないしはろ過等の通常の方法を使用できる。これらの方法により、菌体と母液とに容易にかつ任意の割合に分ける事ができる。培地成分の除去量の割合は、培養液の５０〜９９容量％が好ましく、特に好ましくは７０〜９０容量％である。
【００１７】
培養液から培地成分を一部ないしは大部分を除去した後、微生物含有液に直接あるいは微生物含有液を無機燐酸緩衝液、トリス緩衝液等の緩衝液で希釈したものに、反応基質である、リボース−１−燐酸若しくはその塩及び５−メチルウラシル、または、ヌクレオシド、無機燐酸若しくはその塩及び５−メチルウラシルを添加し、酵素反応を行い、５−メチルウリジンを生成する。反応は、ｐＨ範囲は４〜１０、好ましくは６〜８、温度範囲は、２０〜７０℃、好ましくは５０〜７０℃の条件下、静地あるいは撹拌しながら、１０分〜１０日間行われる。
【００１８】
培養液の培地成分を除去する事無く、培養液にそのまま核酸類の基質を添加した後反応させた場合、得られる結晶の粒度は長径で２０から３０μｍであった。この粒径では、結晶の例えば遠心ろ過を行った場合の分離速度が非常に遅く、大きな装置が必要である事が判明した。
【００１９】
これに対し、培養液の培地成分を一部、または大部分を除去した後に核酸類の基質を添加し、反応を行った場合には、５０から５５０μｍの粒径の結晶の得られる事を発見した。この粒径であれば、結晶の分離速度が速くなり、かつ後述の他の不純物核酸との粒径を利用した分離が可能になる。
【００２０】
発明者は５−メチルウリジンの精製を検討した際、上記の様に、培養液から培地成分を一部ないしは大部分を除去した後基質を加えて反応させると、後工程で得られる５−メチルウリジンの結晶が比較的大きく成る事を発見し、この性質が５−メチルウリジンの精製に活用できうるとの着想を得た。一般的に言って、工業的に粒度の揃った結晶を得ようとする場合、被晶析流体をその流体自体で下方より流動させ、大きな結晶を下部に、細かい結晶を上部に分級させ、上部の細かい結晶を系外で溶解、再循環させる方法が取られるが、本発明のように、製品結晶と不純物結晶を沈降速度で分ける方法は数少ない。その理由は、そのような粒度分布を持った系が無い事、製品結晶中に不純物結晶が取り込まれ、たとえ分ける事ができたとしても純度が上がらない事、および、たとえ分級する事が可能な粒系、結晶の質であっても、発想がそこに至らない為と考えられる。
【００２１】
５−メチルウルジンの場合、市販試薬の結晶粒系は大きくとも５０μｍ程度であったが、発明者がこれを水より再晶析したところ、５００μｍ程の結晶が得られた。
【００２２】
一方、不純物を含む実液系から得られた５−メチルウリジン結晶の粉末Ｘ線回折の結果、５−メチルウリジンは他の不純物と”混晶”を作らず、混合した結晶群の中から５−メチルウリジンの結晶のみを取り出す事ができれば、高純度の製品の得られる事を見いだした。また、実液系での５−メチルウリジンの結晶粒系は通常、３００から６００μｍ、特に小さい場合でも５０から１００μｍの範囲にあり、この時、不純物側の結晶粒系は５から５０μｍであった。さらに、撹判後静置した晶析スラリーの観察により、５−メチルウリジン結晶がガラス容器の下部にあり、上部の液は短時間であれば濁っている事を見いだし、沈降速度の差を使えば、他の結晶を分離できる着想を得た。
【００２３】
具体的に結晶同志を沈降速度で分離する方法としては、一つにはデカンテーション法がある。結晶スラリーをよく混合し、しかる後静置し、５−メチルウリジンが大方沈降し、他の結晶が浮遊してる間に５−メチルウリジン以外の層を容器を傾けて流し去る方法である。流出液を結晶分離し、その母液を元の容器に戻し、再度上記の操作を繰り返す操作を何回か繰り返す事により、不純物結晶を含まない５−メチルウリジン結晶を取得できる。
また、大きな装置では、容器を傾けるのは難しいので、機械力に頼らざるを得ない。たとえば、非沈降層をポンプを用いて吸い出す、あるいは、適当な位置にオーバーフロー口を設けておいて、沈降後、そこから上部の液をオーバーフローさせる等の方法が考えられる。また、一般的な流動層型連続晶析槽を改良し、清澄母液を槽の下部から流し、５−メチルウリジンの結晶は流出させず、不純物結晶のみを流出させる方法、または、いわゆるスーパーデカンター、ハイドロサイクロン等の結晶の分級分離が可能な遠心分離機で一気に、大きな結晶と小さな結晶を分ける方法が考えられる。
【００２４】
デカンテーションないしは上記遠心分離器等を使用する機械的分離を繰り返すことにより、所定の分離の効率と、所定の製品純度を得ることが可能である。
【００２５】
これらのいずれの方法も、分離された不純物結晶を含む流体を遠心分離機、ろ過等で結晶分離し、その母液を元の装置にもどす操作があれば不純物結晶分離効率をより高める事が可能になる。
【００２６】
分離した５−メチルウリジン側の結晶の層を遠心分離、ろ過等で分離し、さらにこの時、結晶を水で、可能ならば冷水で洗浄すれば、より純度の高い結晶が得られる。
【００２７】
５−メチルウリジンを製造するに当たり、核酸を原料とし、微生物を用いて変換する方法の代表例として、ヌクレオシド、無機リン酸および５ーメチルウラシル（チミン）を微生物存在下で作用させる方法がある。ここで、ヌクレオシドとしては、アデノシン、グアノシン、イノシン、ウリジン、シチジンまたはキサントシンが例示される（特開平２−２３８８２）。この反応において、不純物としてそれぞれの残留ヌクレオシド、５ーメチルウラシル（チミン）、使用したヌクレオシドから副成した塩基およびリン酸が存在する。これらの不純物を５−メチルウリジンとの溶解度の差で分離する事は容易に考えられるが、核酸成分の温度、ｐＨに対する溶解度変化のパターンはお互いに類似しており、これ以上分ける事は容易ではない。
【００２８】
この反応液中で、不純物核酸の除去を検討する過程で、発明者は５−メチルウリジンの結晶が比較的大きくなるのに対し、他のものが大きくならない事を見いだした。さらに、５−メチルウリジンの流体中での沈降速度が他の結晶に比べ、著しく大きい事を発見した。
【００２９】
特開平２−２３８８２の条件に加え、培養液から培地成分を一部ないしは大部分を除去した後基質を加えて反応させた後、反応液を冷却、ろ過、活性炭脱色、ろ過して得たろ液を濃縮し、３から１５℃の範囲に冷却晶析して、何等他の溶媒を加える事無く析出してくる結晶のうち、５−メチルウリジン結晶の平均的粒系が５０から５５０μｍであるのに対し、不純物のそれは１０から３０μｍ程度であった。
【００３０】
そこで、流体中の５−メチルウリジン結晶を５分から１０分で沈降させておいた後、５−メチルウリジン結晶層以外の部分を容器を傾けて流出させ（デカンテーション）、さらに流出液中の結晶を分離後、その母液を容器に戻し、撹判後、再びデカンテーションする操作を繰り返したところ、他の結晶をあまり含まない高純度の５−メチルウリジンが得られた。
【００３１】
晶析装置の規模または結晶粒径により、５−メチルウリジンの沈降に要する時間は変化するが、その時の所用時間は、晶泥の一部を抜き取り、メスシリンダー等の中で実際に沈降させ、それに要した時間と沈降距離を計り、これから実際の装置中での所用沈降速度を計算する事により、容易に沈降時間を設定する事ができる。
【００３２】
一方、不純物の結晶粒径は、常にほぼ一定であるから、沈降時間は５−メチルウリジン側だけを考えれば良い。あまり長い時間沈降時間を取ると、不純物結晶も多少沈降してくる。メスシリンダーのような小さな装置では、３０分程度で微結晶の沈降層が形成された。この場合にも、不純物結晶が沈降してくる時間は、装置の規模に基づいて容易に計算でき、沈降時間の限度はこれにより決める事ができる。
【００３３】
流動層型連続晶析槽を改良、あるいは流動層分級型晶析缶を設計して用いる場合には、清澄母液を槽の下部から流し、５−メチルウリジンの結晶は流出させず、不純物結晶のみを流出させるに必要な流体の線速度を測定する事により、容易に実装置での線速度を決定する事ができる。
【００３４】
また同様に、いわゆるスーパーデカンターを用いる場合、小型機を用い、沈降面積あたりの流体フィード流速と結晶の分離性を測定する事により、容易に実機でのフィード流量を設定する事ができる。
【００３５】
ここに、われわれは結晶の沈降速度の差を利用する方法が、５−メチルウリジンと他の核酸成分とを分離する有効な方法である事を見いだした。
【００３６】
上記の方法は、５−メチルウリジンとの分離が極めて難しいチミンの分離に特に大きな効力を発揮した。チミンは溶解度の温度、ｐＨに対する変化のパターンが５−メチルウリジンに相似であり、分離が困難であり、また、イオン解離のパターンも似ており、イオン交換樹脂等でも分離が困難であるため、本発明の方法法は、非常に有効であった。
【００３７】
また、共存する他の核酸であるグアノシン、グアニン、シュドグアノシンについても非常に有効な分離手段であった。
【００３８】
微生物の培養液中の培地成分の一部ないしは大部分を除去した場合にのみ、５−メチルウリジンの大きな粒径の結晶が得られる理由は明確では無いが、酵素反応中に培地成分中に含まれるアミノ酸と他の成分、例えば糖が反応して、その生成物が５−メチルウリジンの結晶の成長を阻害しているのではないかと考えられる。本微生物を用いた反応の温度は６０℃であり、一般的な微生物を用いた生産に比べて温度が高く、この事が前述の副生反応を促進していると考えられ、本特許の培地成分の除去の必要性をもたらしているものと考えられる。
【００３９】
【実施例】
以下、実施例により本発明を更に説明する。
【００４０】
実施例１
特開平２−２３８８２号公報の実施例１に従い、フラボバクテリウム レナナム（Ｆｌａｖｏｂａｃｔｅｒｉｕｍ ｒｈｅｎａｎｕｍ） ＦＥＲＭ ＢＰ−１８６２を培養し、菌体培養液を調製した。この菌体培養液を遠心処理し、培地成分の５０％を除去した。これにトリス緩衝液を加えて元の容量に戻し、基質の５−メチルウラシルとグアノシンを添加し、温度を６０℃に保って反応させた。反応液を冷却、ろ過、活性炭脱色、ろ過、真空濃縮し、これを５℃迄冷却した。得られた晶析スラリー中の結晶の大きさは約８０μｍであった。また、５−メチルウリジンと他の不純物核酸結晶とは自然沈降で良く二層に分離していた。
【００４１】
実施例２
特開平２−２３８８２号公報の実施例１に従い、フラボバクテリウム レナナム（Ｆｌａｖｏｂａｃｔｅｒｉｕｍ ｒｈｅｎａｎｕｍ） ＦＥＲＭ ＢＰ−１８６２を培養し、菌体培養液を調製した。この菌体培養液を遠心処理し、培地成分の９０％を除去した。これにトリス緩衝液を加えて元の容量に戻し、基質の５−メチルウラシルとグアノシンを添加し、本発明の実施例１と同様に反応と後処理を行った。得られた晶析スラリー中の結晶の大きさは約７０μｍであった。また、５−メチルウリジンと他の不純物核酸結晶とは自然沈降で良く二層に分離していた。
【００４２】
実施例３
特開平２−２３８８２号公報の実施例１に従い、フラボバクテリウム レナナム（Ｆｌａｖｏｂａｃｔｅｒｉｕｍ ｒｈｅｎａｎｕｍ） ＦＥＲＭ ＢＰ−１８６２を培養し、菌体培養液を調製した。この菌体培養液を遠心処理し、培地成分の９０％を除去した。これに無機リン酸緩衝液を加えて元の容量に戻し、基質の５−メチルウラシルとグアノシンを添加し、本発明の実施例１と同様に反応と後処理を行った。得られた晶析スラリー中の結晶の大きさは約１００μｍであった。また、５−メチルウリジンと他の不純物核酸結晶とは自然沈降で良く二層に分離していた。
【００４３】
実施例４
実施例１から３の上層を下層の５−メチルウリジン層と上澄み分離し、この下層を遠心分離した所、いずれも速い速度で分離できた。
【００４４】
実施例５
特開平２−２３８８２号公報の実施例２に従い、ミクロコッカス ルテウス（Ｍｉｃｒｏｃｏｃｃｕｓ ｌｕｔｅｕｓ） ＦＥＲＭ Ｐ−７３９９を培養し、菌体培養液を調製後、常法に従い洗浄菌体を得た。これにトリス緩衝液を加えて、元の容量に戻し、基質の５−メチルウラシルとグアノシンを添加し、本発明の実施例１と同様に酵素反応を実施した。得られた酵素反応液を冷却、ろ過、活性炭脱色、ろ過、真空濃縮して、５−メチルウリジン２５．５％、チミン１．９５％、グアノシン０．５７％含む濃縮液３８４．９ｇを得た。これをさらに５０ｒｐｍで撹判しながら徐徐に１０℃まで冷却し、結晶を析出させた。これを１０分間静置し、下部の５−メチルウリジン層以外の上部の微結晶懸濁部分をデカンテーション分離して流出させ、流出液中の結晶をろ過分離し、乾重量あたり５−メチルウリジンを２２．６％、チミンを５０．７％、グアノシンを１０．７％含む８．６２ｇの湿結晶を得た。この母液側を５−メチルウリジン結晶層の残っている元の容器に戻し、撹判し、再び１０℃に冷却した。これを再度、上記と同様にデカンテーション分離し、再び流出液中の結晶をろ過分離し、乾重量あたり５−メチルウリジンを４１．４％、チミンを２６．１％、グアノシンを１４．４％、グアニンを０．０５２％を含む乾燥状態で４．５７ｇの結晶を得た。この母液側を５−メチルウリジン結晶層の残っている元の容器に戻し、撹判しながら１０℃に冷却後、バスケット型遠心ろ過機で流体全量を分離した。分離時に結晶層を５７．８ｇの冷水で結晶を洗浄した。分離結晶を乾燥し、７５．２１ｇの含水和物結晶を得た。濃縮液からの５−メチルウリジンの収率は７１．４％であった。この結晶の純度は９３．２４％であり、不純物として、３．３３％のチミン、０．３４２％のグアノシンが含まれていた。５−メチルウリジンに対するチミンの比率は、濃縮液で７．６％、製品で３．６％であり、晶析、デカンテーション工程で半減していた。更に言えば、定性的であるが、シュドグアノシンも大幅に減少していた。さらに、デカンテーションを繰り返す事により、不純物核酸は更に減少させ得たであろう。なお、この晶析での５−メチルウリジンの結晶平均粒径は約３５０μｍ、微結晶の平均粒径は１０μｍであった。
【００４５】
実施例６
本発明の実施例５と同様にして、菌体培養液を調製後、酵素反応を実施した。この酵素反応液を冷却、ろ過、活性炭脱色、ろ過、真空濃縮して、５−メチルウリジン２３．０％、チミン１．７１％、グアノシン０．４５％含む濃縮液５８０Ｌを得た。これを晶析缶中で１００ｒｐｍで撹判しながら徐徐に５℃まで冷却し、結晶を析出させた。撹判を停止し、約１５分間、５−メチルウリジン結晶を沈降させた後、微結晶を含んだ上部微結晶懸濁液をポンプを用いて機械的に分離した。この分離液中の微結晶をろ過分離し、この母液側を元の晶析缶に戻し、撹判し、再び５℃に冷却した。上記の撹判、静置、上部微結晶懸濁液分離、微結晶ろ過分離、再懸濁の一連の操作を４度繰り返した後、バスケット型遠心ろ過機で懸濁液全体を分離した。分離時に６０Ｌの水で結晶を洗浄した。分離結晶を乾燥し、９６Ｋｇの含水和物結晶を得た。濃縮液からの５−メチルウリジンの収率は７２．０％であった。この結晶の純度は９３．８％であり、不純物として、１．８％のチミン、０．１６％のグアノシンが含まれていた。５−メチルウリジンに対するチミンの比率は、濃縮液で７．４％、製品で１．９％であり、晶析、デカンテーション工程で４分の１に減少していた。また、シュドグアノシンも大幅に減少していた。さらに、デカンテーションを繰り返す事により、不純物核酸は更に減少させ得たであろう。なお、この晶析での５−メチルウリジンの結晶平均粒径は約５５０μｍ、微結晶の平均粒径は１５μｍであった。
【００４６】
比較例１
本発明の実施例１と同様にして、フラボバクテリウム レナナム（Ｆｌａｖｏｂａｃｔｅｒｉｕｍ ｒｈｅｎａｎｕｍ） ＦＥＲＭ ＢＰ−１８６２の菌体培養液を調製した。この菌体培養液の培地成分を除去することなく、基質の５−メチルウラシルとグアノシンを添加し、本発明の実施例１と同様に温度を６０℃に保って反応させた。反応液を冷却、ろ過、活性炭脱色、ろ過、真空濃縮し、これを５℃迄冷却した。得られた５−メチルウリジンの結晶の大きさは２０から３０μｍであり、他の不純物核酸結晶との自然沈降による分離現象は見られなかった。また、遠心分離に非常に長時間要した。
【００４７】
比較例２
本発明の実施例５と同様にして、ミクロコッカス ルテウス（Ｍｉｃｒｏｃｏｃｃｕｓ ｌｕｔｅｕｓ） ＦＥＲＭ Ｐ−７３９９の菌体培養液を調製後、酵素反応を実施した。得られた酵素反応液を冷却、ろ過、活性炭脱色、ろ過、真空濃縮して、５−メチルウリジン２５．２％、チミン１．５５％、グアノシン０．４２％含む濃縮液７．０５ｋｇを得た。これをさらに撹判しながら徐徐に５℃まで冷却し、結晶を析出させた。この懸濁液を静置することなく、すぐにバスケット型遠心ろ過機で全量分離し、５−メチルウリジンを４．５％、チミンを０．２６％、グアノシンを０．１２％含む５２２３ｇの母液を得た。更に結晶層を８１４ｇの冷水で洗浄した。この洗浄時の母液は、５−メチルウリジンを５．１８％、チミンを０．３１％、グアノシンを０．１９％含み、１９２６ｇあった。分離結晶を乾燥し、１．５２ｋｇの含水和物結晶を得た。濃縮液からの５−メチルウリジンの収率は７６．３％であった。この結晶の純度は８９．２８％であり、不純物として、５．２１％のチミン、０．６９％のグアノシンが含まれていた。５−メチルウリジンに対するチミンの比率は、濃縮液で６．２％、製品で５．８％であり、晶析工程での減少は全くなかった。
【００４８】
【発明の効果】
産業上有用な５−メチルウリジンを、有機溶剤等特殊な溶媒を用いる事無く、かつ、簡便な方法で高純度に精製できる。[0001]
[Industrial applications]
The present invention relates to a method for producing 5-methyluridine, which is useful as a pharmaceutical raw material. 5-Methyluridine is promising as a synthetic intermediate for azidothymidine, which is commercially available as an AIDS drug, and also for d4T (2 ', 3'-dihydroxy-2', 3'-dideoxythymidine) which is also in clinical trials.
[0002]
[Prior art]
Conventionally, 5-methyluridine has been produced by a chemical synthesis method, and the purification there has been performed mainly by crystallization using an organic solvent such as methanol or ethanol. (J. Am. Chem. Soc., 78, 2117 (1956); JP-A-63-63668; Helvetica Chimica Acta, 2179 (1980); Synthesis, 259 (1982)).
[0003]
These techniques cannot be referred to as a method for producing 5-methyluridine, including purification, because impurities produced as by-products are different from the method of converting nucleic acid as a raw material and using a microorganism.
[0004]
As a method for producing 5-methyluridine using a microorganism, a method for producing 5-methyluridine by reacting nucleoside or ribose-1-phosphate with 5-methyluracil in the presence of a microorganism is known. (JP-A-2-23882)
[0005]
However, this patent has no specific description on a method for purifying 5-methyluridine, which is insufficient for producing 5-methyluridine as a product.
[0006]
In particular, obtaining large crystals of 5-methyluridine by crystallization can improve the separability of the crystals, reduce the size of the apparatus required for the separation, and improve the separation of impurities from the crystal. Necessary for obtaining a product of purity, but nothing is said about it.
[0007]
Usually, the size of supersaturation and impurities have a great influence on crystal growth, and in particular, the composition of impurities depends entirely on the system to be crystallized, and this control is an important key that determines the possibility of crystallization, Characteristic control in a given system is often required.
[0008]
Therefore, in the case of 5-methyluridine using microorganisms, information necessary for biochemical production is known, but for purification from a reaction solution, it was necessary to independently develop a method.
[0009]
When 5-methyluridine was crystallized from the reaction solution through some pretreatment and concentration, only crystals having a size of 20 to 30 micrometers (μm) were obtained. The device turned out to be a big thing. Further, it has been found that this size is completely inappropriate for the separation of impurities using the difference in crystal grain size described later.
[0010]
5-Methyluridine can be produced by an enzymatic reaction using nucleic acids as a raw material by using microorganisms. At that time, unreacted nucleic acids and by-product nucleic acids are contained in the reaction product. Included as When 5-methyluracil (thymine) and guanosine are used as raw materials for producing 5-methyluridine, impurities in the reaction solution are mainly thymine, guanine, guanosine, and 2-Amino-7-β-D-ribofuranosyl. -7H-purine-6 (1H) one (pseudoguanosine) is present. Among them, for example, thymine is difficult to remove by crystallization because the pattern of change in solubility with respect to temperature, pH and the like is similar to that of 5-methyluridine. Further, since the ion dissociation patterns are similar, it is difficult to remove them by treatment with an ion exchange resin or the like.
[0011]
These nucleic acid-based impurities are peculiar to the method of producing microorganisms using nucleic acids as raw materials, and thus cannot use known knowledge. In addition, the known literature does not describe a method for separating these.
[0012]
[Problems to be solved by the invention]
Efficiently produce 5-methyluridine, and increase the particle size of 5-methyluridine crystals from the reaction solution using microorganisms to increase the efficiency of separation of crystals and mother liquor so that the product becomes highly pure. A method for improving the separation efficiency between this crystal and another impurity crystal is developed.
[0013]
[Means for Solving the Problems]
The inventor cultured a microorganism, produced 5-methyluridine by an enzymatic reaction using nucleic acids as a raw material, and studied a crystallization and separation method. -It has been found that there is a great difference in the particle size of crystals obtained by crystallization of methyluridine, and the present invention has been completed. That is, the present invention relates to a microorganism capable of producing 5-methyluridine from ribose-1-phosphate or a salt thereof and 5-methyluracil, or a nucleoside, an inorganic phosphate or a salt thereof, and 5-methyluridine from 5-methyluracil. In a method for producing 5-methyluridine by an enzymatic reaction using a microorganism having an ability to produce, after culturing the microorganism,
(A) removing part or all of the medium components; and (b) adding to the microorganism ribose-1-phosphate or a salt thereof and 5-methyluracil or nucleoside, an inorganic phosphate or a salt thereof and 5-methyluracil. The present invention relates to a method for producing 5-methyluridine, which comprises reacting and producing 5-methyluridine, and then (c) crystallizing and separating the produced 5-methyluridine.
[0014]
The microorganism used in the present invention is a microorganism capable of producing 5-methyluridine from ribose-1-phosphate or a salt thereof and 5-methyluracil, or a nucleoside, an inorganic phosphate or a salt thereof, and 5-methyluracil from 5-methyluracil. Any microorganism capable of producing uridine can be used. For example, microorganisms described in Patent Publication JP-A-2-23882 such as Achromobacter, Acinetobacter, Aeromonas and the like can be mentioned. Particularly preferred microorganisms include microorganisms belonging to the genera Arthrobacter, Cellulomonas, Flavobacterium, Klebjella, Microbacterium, Micrococcus, and Sarsina. Specific examples thereof include the following microorganisms.
Arthrobacter simplex FERM P-10068
Cellulomonas flavigena ATCC 486
Flavobacterium rhenanum FERM BP-1862
Klebsiella pneumoniae ATCC 8308
Microbacterium lacticum ATCC 8180
Micrococcus luteus FERM P-7399
Sarcina lutea FERM P-7400
[0015]
The culture of the microorganism contains a carbon source, a nitrogen source, inorganic ions such as P, S, Fe, and Mn, and if necessary, a micronutrient or a protein hydrolyzate such as a vitamin, and an organic nitrogen source such as a yeast extract. What is necessary is just to perform by a normal culture method using a normal culture medium.
[0016]
As a method for removing the medium component from the culture solution of the obtained microorganism, a usual method such as removal of the supernatant after natural sedimentation of the culture solution, centrifugation or filtration can be used. By these methods, the cells can be easily divided into the cells and the mother liquor at an arbitrary ratio. The ratio of the removal amount of the medium component is preferably 50 to 99% by volume of the culture solution, and particularly preferably 70 to 90% by volume.
[0017]
After removing some or most of the medium components from the culture solution, the reaction substrate, ribose, is directly added to the microorganism-containing solution or diluted with a buffer solution such as an inorganic phosphate buffer or Tris buffer. -1-Phosphoric acid or a salt thereof and 5-methyluracil, or nucleoside, inorganic phosphoric acid or a salt thereof and 5-methyluracil are added, and an enzymatic reaction is carried out to produce 5-methyluridine. The reaction is carried out under the conditions of a pH range of 4 to 10, preferably 6 to 8 and a temperature range of 20 to 70 ° C., preferably 50 to 70 ° C., for 10 minutes to 10 days while stirring or under stirring.
[0018]
When the reaction was performed after the nucleic acid substrate was directly added to the culture solution without removing the medium components of the culture solution, the resulting crystals had a long diameter of 20 to 30 μm. With this particle size, it has been found that the separation speed when, for example, centrifugal filtration of the crystals is performed is very slow, and a large apparatus is required.
[0019]
On the other hand, it has been found that when a part of or most of the medium component of the culture solution is removed and a nucleic acid substrate is added and the reaction is performed, crystals having a particle size of 50 to 550 μm can be obtained. did. With this particle size, the separation speed of the crystal is increased, and separation using the particle size with other impurity nucleic acids described later becomes possible.
[0020]
When the inventor examined the purification of 5-methyluridine, as described above, if a part or most of the medium component was removed from the culture solution, and a substrate was added thereto, and the reaction was carried out, the 5-methyluridine obtained in the subsequent step was obtained. They discovered that uridine crystals were relatively large and inspired that this property could be used for the purification of 5-methyluridine. Generally speaking, when trying to obtain crystals of uniform grain size industrially, the fluid to be crystallized is flowed from below by the fluid itself, large crystals are classified into the lower part, and fine crystals are classified into the upper part. A method of dissolving and recirculating fine crystals outside the system is used, but there are few methods of separating product crystals and impurity crystals at a sedimentation rate as in the present invention. The reason is that there is no system with such a particle size distribution, impurity crystals are incorporated into product crystals, and even if they can be separated, the purity does not increase, and it is possible to classify This is probably because even the quality of the grain system or the crystal does not reach the idea.
[0021]
In the case of 5-methylurdin, the crystal grain system of the commercially available reagent was at most about 50 μm. When the inventor recrystallized this from water, crystals of about 500 μm were obtained.
[0022]
On the other hand, as a result of powder X-ray diffraction of 5-methyluridine crystal obtained from a real liquid system containing impurities, 5-methyluridine did not form “mixed crystal” with other impurities, but was found to be 5% of the mixed crystal group. -It has been found that if only methyluridine crystals can be taken out, a product of high purity can be obtained. Further, the crystal grain system of 5-methyluridine in the actual liquid system is usually in the range of 300 to 600 μm, particularly in the range of 50 to 100 μm even in a small case, and the crystal grain system on the impurity side is 5 to 50 μm. . Furthermore, by observing the crystallization slurry that was allowed to stand after stirring, it was found that the 5-methyluridine crystal was at the bottom of the glass container, and the liquid at the top was turbid for a short time. For example, I got the idea of separating other crystals.
[0023]
As a specific method for separating crystals at a sedimentation velocity, there is a decantation method. This is a method in which the crystal slurry is mixed well and then allowed to stand, and while 5-methyluridine has largely settled and other crystals are floating, the layers other than 5-methyluridine are tilted off and washed away. The effluent is subjected to crystal separation, the mother liquor is returned to the original container, and the above operation is repeated several times to obtain a 5-methyluridine crystal containing no impurity crystal.
In addition, in a large device, it is difficult to tilt the container, so that it is necessary to rely on mechanical force. For example, a method of sucking out a non-settling layer using a pump, or providing an overflow port at an appropriate position, and allowing the liquid in the upper portion to overflow from the settling port after the settling is considered. In addition, a general fluidized bed continuous crystallization tank is improved, a clarified mother liquor is allowed to flow from the bottom of the tank, and 5-methyluridine crystals are not allowed to flow out, but only impurity crystals are allowed to flow out, or a so-called super decanter, A method of separating large crystals and small crystals at once using a centrifuge capable of classifying and separating crystals such as hydrocyclones is conceivable.
[0024]
By repeating decantation or mechanical separation using the above-mentioned centrifugal separator or the like, it is possible to obtain a predetermined separation efficiency and a predetermined product purity.
[0025]
In any of these methods, if there is an operation of separating the fluid containing the separated impurity crystals by a centrifugal separator, filtration, etc., and returning the mother liquor to the original device, it is possible to further increase the impurity crystal separation efficiency. Become.
[0026]
The separated crystal layer on the 5-methyluridine side is separated by centrifugation, filtration and the like, and at this time, the crystals are washed with water and, if possible, with cold water, to thereby obtain crystals with higher purity.
[0027]
In producing 5-methyluridine, as a typical example of a method of converting a nucleic acid as a raw material using a microorganism, there is a method in which nucleoside, inorganic phosphate, and 5-methyluracil (thymine) are allowed to act in the presence of the microorganism. Here, examples of the nucleoside include adenosine, guanosine, inosine, uridine, cytidine and xanthosine (JP-A-2-23882). In this reaction, each residual nucleoside, 5-methyluracil (thymine), a base formed from the used nucleoside and phosphoric acid are present as impurities. It is easy to separate these impurities by the difference in solubility with 5-methyluridine, but the patterns of solubility change of nucleic acid components with respect to temperature and pH are similar to each other, and it is not easy to separate them further. Absent.
[0028]
In the course of examining the removal of the impurity nucleic acid in this reaction solution, the inventor found that the crystals of 5-methyluridine were relatively large while the others were not. Further, they have found that the sedimentation rate of 5-methyluridine in a fluid is significantly higher than that of other crystals.
[0029]
In addition to the conditions described in JP-A-2-23882, after removing a part or most of the medium component from the culture solution, a substrate is added and reacted, and then the reaction solution is cooled, filtered, decolorized with activated carbon, and filtrate obtained by filtration. Is concentrated and crystallized by cooling to a temperature in the range of 3 to 15 ° C., and among the crystals precipitated without adding any other solvent, the average grain system of 5-methyluridine crystals is 50 to 550 μm. On the other hand, that of the impurity was about 10 to 30 μm.
[0030]
Therefore, after the 5-methyluridine crystals in the fluid are allowed to settle in 5 to 10 minutes, portions other than the 5-methyluridine crystal layer are tilted out of the vessel (decantation), and the crystals in the effluent are further decanted. After the separation, the mother liquor was returned to the container, and after stirring, the operation of decanting was repeated again, whereby high-purity 5-methyluridine containing little other crystals was obtained.
[0031]
The time required for sedimentation of 5-methyluridine varies depending on the scale or crystal grain size of the crystallizer, but the time required at that time is to extract a part of the crystal mud and allow it to actually settle in a graduated cylinder, etc. The required settling time can be easily set by measuring the required time and the settling distance and calculating the required settling velocity in the actual apparatus.
[0032]
On the other hand, since the crystal grain size of the impurities is almost always constant, the sedimentation time may be considered only on the 5-methyluridine side. If the sedimentation time is too long, the impurity crystals will also slightly settle. With a small device such as a measuring cylinder, a sedimented layer of microcrystals was formed in about 30 minutes. Also in this case, the time during which the impurity crystals settle can be easily calculated based on the scale of the apparatus, and the limit of the settling time can be determined thereby.
[0033]
When improving a fluidized bed type continuous crystallization tank or designing and using a fluidized bed classification type crystallizer, clarify the mother liquor from the bottom of the tank, do not allow 5-methyluridine crystals to flow out, and only impurity crystals. By measuring the linear velocity of the fluid required to allow the fluid to flow out, the linear velocity in the actual device can be easily determined.
[0034]
Similarly, when a so-called super decanter is used, the feed flow rate in the actual machine can be easily set by using a small machine and measuring the fluid feed flow rate per sedimentation area and the separability of crystals.
[0035]
Here, we have found that a method utilizing the difference in the sedimentation rates of the crystals is an effective method for separating 5-methyluridine from other nucleic acid components.
[0036]
The above method was particularly effective for separating thymine, which is extremely difficult to separate from 5-methyluridine. Thymine has a solubility pattern similar to that of 5-methyluridine in the pattern of change with respect to temperature and pH, and is difficult to separate.In addition, the pattern of ion dissociation is also similar, and it is difficult to separate even an ion exchange resin. The method of the present invention was very effective.
[0037]
In addition, guanosine, guanine, and pseudoguanosine, which are other coexisting nucleic acids, were also very effective separation means.
[0038]
Although it is not clear why crystals having a large particle size of 5-methyluridine can be obtained only when a part or most of the medium component in the culture solution of the microorganism is removed, it is not included in the medium component during the enzyme reaction. It is thought that the amino acid to be reacted with another component, for example, a sugar, and the product inhibits the growth of crystals of 5-methyluridine. The temperature of the reaction using the present microorganism is 60 ° C., which is higher than the production using a general microorganism, which is considered to promote the by-product reaction described above. It is believed that this has necessitated the removal of components.
[0039]
【Example】
Hereinafter, the present invention will be further described with reference to examples.
[0040]
Example 1
According to Example 1 of JP-A-2-23882, Flavobacterium rhenanum FERM BP-1862 was cultured to prepare a cell culture solution. The cell culture was centrifuged to remove 50% of the medium components. To this, Tris buffer was added to return to the original volume, the substrates 5-methyluracil and guanosine were added, and the reaction was carried out while maintaining the temperature at 60 ° C. The reaction solution was cooled, filtered, decolorized with activated carbon, filtered and concentrated in vacuo, and cooled to 5 ° C. The size of the crystals in the obtained crystallization slurry was about 80 μm. In addition, 5-methyluridine and other impurity nucleic acid crystals were well separated by natural sedimentation into two layers.
[0041]
Example 2
According to Example 1 of JP-A-2-23882, Flavobacterium rhenanum FERM BP-1862 was cultured to prepare a cell culture solution. The cell culture was centrifuged to remove 90% of the medium components. To this, Tris buffer was added to return to the original volume, and the substrates 5-methyluracil and guanosine were added, and the reaction and post-treatment were carried out in the same manner as in Example 1 of the present invention. The size of the crystals in the obtained crystallization slurry was about 70 μm. In addition, 5-methyluridine and other impurity nucleic acid crystals were well separated by natural sedimentation into two layers.
[0042]
Example 3
According to Example 1 of JP-A-2-23882, Flavobacterium rhenanum FERM BP-1862 was cultured to prepare a cell culture solution. The cell culture was centrifuged to remove 90% of the medium components. An inorganic phosphate buffer was added thereto to return the volume to the original volume, and 5-methyluracil and guanosine as substrates were added, and the reaction and post-treatment were carried out in the same manner as in Example 1 of the present invention. The size of the crystals in the obtained crystallization slurry was about 100 μm. In addition, 5-methyluridine and other impurity nucleic acid crystals were well separated by natural sedimentation into two layers.
[0043]
Example 4
The upper layer of each of Examples 1 to 3 was separated from the lower 5-methyluridine layer by supernatant, and the lower layer was centrifuged.
[0044]
Example 5
According to Example 2 of JP-A-2-23882, Micrococcus luteus FERM P-7399 was cultured, and a cell culture solution was prepared, and washed cells were obtained according to a conventional method. To this was added Tris buffer to return to the original volume, and the substrates 5-methyluracil and guanosine were added, and the enzyme reaction was carried out in the same manner as in Example 1 of the present invention. The obtained enzyme reaction solution was cooled, filtered, decolorized with activated carbon, filtered, and concentrated in vacuo to obtain 384.9 g of a concentrated solution containing 25.5% of 5-methyluridine, 1.95% of thymine, and 0.57% of guanosine. . The mixture was gradually cooled to 10 ° C. while stirring at 50 rpm to precipitate crystals. This was allowed to stand for 10 minutes, the upper part of the microcrystal suspension other than the lower 5-methyluridine layer was decanted and allowed to flow, and the crystals in the effluent were separated by filtration, and 5-methyluridine per dry weight was obtained. 8.62 g of wet crystals containing 22.6%, thymine 50.7% and guanosine 10.7% were obtained. The mother liquor side was returned to the original container in which the 5-methyluridine crystal layer remained, stirred, and cooled again to 10 ° C. This was again decanted and separated in the same manner as above, and the crystals in the effluent were again separated by filtration. Based on the dry weight, 5-methyluridine was 41.4%, thymine was 26.1%, and guanosine was 14.4%. In a dry state containing 0.052% of guanine, 4.57 g of crystals were obtained. This mother liquor side was returned to the original container in which the 5-methyluridine crystal layer remained, cooled to 10 ° C. while stirring, and the entire amount of the fluid was separated by a basket-type centrifugal filter. At the time of separation, the crystal layer was washed with 57.8 g of cold water. The separated crystals were dried to obtain 75.21 g of hydrate crystals. The yield of 5-methyluridine from the concentrate was 71.4%. The purity of the crystal was 93.24%, and contained 3.33% of thymine and 0.342% of guanosine as impurities. The ratio of thymine to 5-methyluridine was 7.6% for the concentrate and 3.6% for the product, and was reduced by half in the crystallization and decantation steps. Furthermore, qualitatively, pseudoguanosine was also significantly reduced. Further, by repeating the decantation, the impurity nucleic acid could have been further reduced. In this crystallization, the average crystal grain size of 5-methyluridine was about 350 μm, and the average crystal grain size was 10 μm.
[0045]
Example 6
In the same manner as in Example 5 of the present invention, an enzyme reaction was performed after preparing a bacterial cell culture solution. The enzymatic reaction solution was cooled, filtered, decolorized with activated carbon, filtered and concentrated in vacuo to obtain 580 L of a concentrated solution containing 23.0% of 5-methyluridine, 1.71% of thymine and 0.45% of guanosine. This was gradually cooled to 5 ° C. while stirring at 100 rpm in a crystallizer to precipitate crystals. After stopping the stirring and sedimenting the 5-methyluridine crystals for about 15 minutes, the upper microcrystal suspension containing the microcrystals was separated mechanically using a pump. The fine crystals in the separated solution were separated by filtration, and the mother liquor side was returned to the original crystallizer, stirred, and cooled again to 5 ° C. After repeating a series of the above operations of stirring, standing, separation of the upper microcrystal suspension, separation of microcrystal filtration, and resuspension four times, the entire suspension was separated by a basket-type centrifugal filter. At the time of separation, the crystals were washed with 60 L of water. The separated crystals were dried to obtain hydrate crystals of 96 kg. The yield of 5-methyluridine from the concentrate was 72.0%. The purity of the crystals was 93.8%, and contained 1.8% thymine and 0.16% guanosine as impurities. The ratio of thymine to 5-methyluridine was 7.4% in the concentrate and 1.9% in the product, and was reduced by a factor of 4 in the crystallization and decantation steps. In addition, pseudoguanosine also decreased significantly. Further, by repeating the decantation, the impurity nucleic acid could have been further reduced. In this crystallization, the average crystal particle size of 5-methyluridine was about 550 μm, and the average particle size of the fine crystals was 15 μm.
[0046]
Comparative Example 1
In the same manner as in Example 1 of the present invention, a cell culture broth of Flavobacterium rhenanum FERM BP-1862 was prepared. Without removing the medium component of the cell culture solution, substrates 5-methyluracil and guanosine were added, and the reaction was carried out at a temperature of 60 ° C. as in Example 1 of the present invention. The reaction solution was cooled, filtered, decolorized with activated carbon, filtered and concentrated in vacuo, and cooled to 5 ° C. The size of the obtained 5-methyluridine crystals was 20 to 30 μm, and no separation phenomenon by spontaneous sedimentation with other impurity nucleic acid crystals was observed. In addition, centrifugation took a very long time.
[0047]
Comparative Example 2
A bacterial culture of Micrococcus luteus FERM P-7399 was prepared in the same manner as in Example 5 of the present invention, and then an enzyme reaction was performed. The obtained enzyme reaction solution was cooled, filtered, decolorized with activated carbon, filtered, and concentrated in vacuo to obtain 7.05 kg of a concentrated solution containing 55.2% of 5-methyluridine, 1.55% of thymine, and 0.42% of guanosine. . This was gradually cooled to 5 ° C. with further stirring to precipitate crystals. Without suspension, the whole amount was immediately separated by a basket centrifugal filter, and 5223 g of a mother liquor containing 4.5% of 5-methyluridine, 0.26% of thymine and 0.12% of guanosine was used. I got Further, the crystal layer was washed with 814 g of cold water. The mother liquor at the time of the washing contained 5.926% of 5-methyluridine, 0.31% of thymine and 0.19% of guanosine, and weighed 1926 g. The separated crystals were dried to obtain 1.52 kg of hydrate crystals. The yield of 5-methyluridine from the concentrate was 76.3%. The purity of the crystal was 89.28%, and contained 5.21% of thymine and 0.69% of guanosine as impurities. The ratio of thymine to 5-methyluridine was 6.2% in the concentrate and 5.8% in the product, and there was no decrease in the crystallization step.
[0048]
【The invention's effect】
Industrially useful 5-methyluridine can be purified to a high purity by a simple method without using a special solvent such as an organic solvent.
Claims (4)
（イ） 該培地成分の一部、または全部を除去し、次いで
（ロ） 該微生物にリボース一燐酸若しくはその塩及び５−メチルウラシルまたはヌクレオシド、無機燐酸若しくはその塩及び５−メチルウラシルを作用せしめ、５−メチルウリジンを生成させた後、次いで
（ハ） 生成した5−メチルウリジンを晶析し、分離する
ことを特徴とする５−メチルウリジンの製造方法A microorganism capable of producing 5-methyluridine from ribose-1-phosphate or a salt thereof and 5-methyluracil or a microorganism capable of producing 5-methyluridine from a nucleoside, an inorganic phosphate or a salt thereof, and 5-methyluracil In the method for producing 5-methyluridine by enzymatic reaction, after culturing the microorganism,
(A) removing part or all of the medium components, and (b) reacting the microorganism with ribose monophosphate or a salt thereof and 5-methyluracil or nucleoside, an inorganic phosphate or a salt thereof and 5-methyluracil. , 5-methyluridine is produced, and then (c) the produced 5-methyluridine is crystallized and separated.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9286195A JP3586924B2 (en) | 1994-04-18 | 1995-04-18 | Method for producing 5-methyluridine |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6-78783 | 1994-04-18 | ||
| JP7878394 | 1994-04-18 | ||
| JP9286195A JP3586924B2 (en) | 1994-04-18 | 1995-04-18 | Method for producing 5-methyluridine |
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| Publication Number | Publication Date |
|---|---|
| JPH08282A JPH08282A (en) | 1996-01-09 |
| JP3586924B2 true JP3586924B2 (en) | 2004-11-10 |
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| JP9286195A Expired - Fee Related JP3586924B2 (en) | 1994-04-18 | 1995-04-18 | Method for producing 5-methyluridine |
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| US6015796A (en) * | 1998-03-11 | 2000-01-18 | Zeria Pharmaceutical Co., Ltd. | Method for treating AIDS |
| JP5448588B2 (en) * | 2009-06-12 | 2014-03-19 | 株式会社トクヤマ | Method for purifying L-carnosine |
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