JPH01193280A - Concentration and purification of antibiotic - Google Patents
Concentration and purification of antibioticInfo
- Publication number
- JPH01193280A JPH01193280A JP1793888A JP1793888A JPH01193280A JP H01193280 A JPH01193280 A JP H01193280A JP 1793888 A JP1793888 A JP 1793888A JP 1793888 A JP1793888 A JP 1793888A JP H01193280 A JPH01193280 A JP H01193280A
- Authority
- JP
- Japan
- Prior art keywords
- antibiotic
- temperature
- phase separation
- solvents
- solution
- 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.)
- Granted
Links
- 230000003115 biocidal effect Effects 0.000 title claims abstract description 29
- 238000000746 purification Methods 0.000 title description 8
- 238000005191 phase separation Methods 0.000 claims abstract description 24
- 239000003960 organic solvent Substances 0.000 claims abstract description 13
- 239000000126 substance Substances 0.000 claims abstract description 9
- 239000002253 acid Substances 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 26
- 239000003242 anti bacterial agent Substances 0.000 claims description 15
- 229940088710 antibiotic agent Drugs 0.000 claims description 13
- 150000003839 salts Chemical class 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 2
- 230000008014 freezing Effects 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims 1
- 244000005700 microbiome Species 0.000 claims 1
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 abstract description 24
- 239000012141 concentrate Substances 0.000 abstract description 6
- 239000002244 precipitate Substances 0.000 abstract description 5
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 abstract description 4
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 abstract description 4
- 239000013543 active substance Substances 0.000 abstract description 2
- 229940009456 adriamycin Drugs 0.000 abstract description 2
- 239000002904 solvent Substances 0.000 abstract 4
- SFENEGWBRXYBJT-UHFFFAOYSA-N 2-methylpropanoic acid;hydrate Chemical compound O.CC(C)C(O)=O SFENEGWBRXYBJT-UHFFFAOYSA-N 0.000 abstract 1
- YMZJBJPWTXJQMR-LJUKVTEVSA-L Fosfomycin calcium Chemical compound [Ca+2].C[C@@H]1O[C@@H]1P([O-])([O-])=O YMZJBJPWTXJQMR-LJUKVTEVSA-L 0.000 abstract 1
- MJZLMKCSLOOMPN-UHFFFAOYSA-N butanoic acid;hydrate Chemical compound O.CCCC(O)=O MJZLMKCSLOOMPN-UHFFFAOYSA-N 0.000 abstract 1
- 230000001112 coagulating effect Effects 0.000 abstract 1
- 238000002844 melting Methods 0.000 abstract 1
- 230000008018 melting Effects 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
- 239000006228 supernatant Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 20
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 239000012071 phase Substances 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 238000000605 extraction Methods 0.000 description 7
- 239000007788 liquid Substances 0.000 description 6
- 238000001816 cooling Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 4
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 3
- WEAHRLBPCANXCN-UHFFFAOYSA-N Daunomycin Natural products CCC1(O)CC(OC2CC(N)C(O)C(C)O2)c3cc4C(=O)c5c(OC)cccc5C(=O)c4c(O)c3C1 WEAHRLBPCANXCN-UHFFFAOYSA-N 0.000 description 3
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 229960004857 mitomycin Drugs 0.000 description 2
- 235000010344 sodium nitrate Nutrition 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 1
- 108010001478 Bacitracin Proteins 0.000 description 1
- DYDCUQKUCUHJBH-UWTATZPHSA-N D-Cycloserine Chemical compound N[C@@H]1CONC1=O DYDCUQKUCUHJBH-UWTATZPHSA-N 0.000 description 1
- DYDCUQKUCUHJBH-UHFFFAOYSA-N D-Cycloserine Natural products NC1CONC1=O DYDCUQKUCUHJBH-UHFFFAOYSA-N 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- 241001647006 Myxococcus virescens Species 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 1
- 229960003942 amphotericin b Drugs 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229940045799 anthracyclines and related substance Drugs 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003972 antineoplastic antibiotic Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 229960003071 bacitracin Drugs 0.000 description 1
- 229930184125 bacitracin Natural products 0.000 description 1
- CLKOFPXJLQSYAH-ABRJDSQDSA-N bacitracin A Chemical compound C1SC([C@@H](N)[C@@H](C)CC)=N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(=O)N[C@H](CCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2N=CNC=2)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCCCC1 CLKOFPXJLQSYAH-ABRJDSQDSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 239000012045 crude solution Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 229960003077 cycloserine Drugs 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229960000308 fosfomycin Drugs 0.000 description 1
- YMDXZJFXQJVXBF-STHAYSLISA-N fosfomycin Chemical compound C[C@@H]1O[C@@H]1P(O)(O)=O YMDXZJFXQJVXBF-STHAYSLISA-N 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 108010082754 iturin A Proteins 0.000 description 1
- -1 lactam lactone Chemical class 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000010687 lubricating oil Substances 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 238000011403 purification operation Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000000956 solid--liquid extraction Methods 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 210000001179 synovial fluid Anatomy 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、相分離法による抗生物質の新規濃縮精製方法
に関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel method for concentrating and purifying antibiotics by phase separation method.
抗生物質の抽出技術としては、通常の固液抽出のほか、
抽出当初より二相系になっている溶媒抽出などが周知で
ある。ところが、抽出開始時は均一相系でありながら温
度変化により多相系にする技法（以下、「相分離法」と
いう）全抗生物質の抽出・濃縮に応用した例は知られて
いなかった。In addition to conventional solid-liquid extraction, antibiotic extraction techniques include
Solvent extraction, which is a two-phase system from the beginning of extraction, is well known. However, there was no known example of applying this technique, which is a homogeneous phase system at the start of extraction but changes to a multiphase system by temperature changes (hereinafter referred to as "phase separation method"), to the extraction and concentration of whole antibiotics.
過去には、パラフィン系潤滑油やナフテン系油を相分Ｎ
ｋ法により選択的に抽出する試みがなされた程度であっ
た。（Ｆｒａｎｅｉ＠Ａ、Ｗｏ、Ｃｒ１ｔｉｃａｌＳｏ
ｌｕｔｉｏｎ Ｔｅｍｐｅｒａｔｕｒ＠ＩＩ、Ａｍｅｒ
ｉｅａｎ ＣｈｅｍｉｃａｌＳｏｃｌｅｔｙ（１９６１
））
また、抗生物質の凝縮又は精製操作については、通常、
沈殿法９分散吸着法、凍結乾燥法、減圧濃縮法等がよく
知られている。そして、有機溶媒の除去には、上記鏝縮
操作で：ＡＩｆ？、されるものもあるが、これらの方法
は、目的とする抗生物質の回収を低下させたり操作その
ものの負荷が大きい為に、煩雑な場合が生じ、注意會要
するものであった。In the past, paraffin-based lubricating oils and naphthenic oils were
Only attempts were made to selectively extract them using the k method. (Franei@A,Wo,CrlticalSo
lution Temperature@II, Amer
ian Chemical Society (1961
)) Also, regarding condensation or purification operations of antibiotics,
Precipitation methods 9 Dispersion adsorption methods, freeze drying methods, vacuum concentration methods, etc. are well known. Then, to remove the organic solvent, use the above troweling operation: AIf? However, since these methods reduce the recovery of the target antibiotic and require a large operational load, they are sometimes complicated and require careful attention.
抗生物質を簡便かつ効率的に濃縮精製する工業的方法の
開発が望まれている。There is a need for the development of an industrial method for concentrating and purifying antibiotics simply and efficiently.
本発明者らは上記課題を解決すべく鋭意検討した結果、
抗生物質金相分離法により濃縮精製する方法を見出し、
この発見に基き本発明を完成するに到った。As a result of intensive study by the present inventors to solve the above problems,
Discovered a method for concentrating and purifying antibiotics using gold phase separation method.
Based on this discovery, we have completed the present invention.
即ち、不発ＦＪＡは、温度変化により相分離する混合有
機溶媒の抗生物質含有溶液を温度変化により相分離せし
めることを特徴とする抗生物質の濃縮精良方法に関する
ものである。That is, the unexploded FJA relates to a method for concentrating and purifying antibiotics, which is characterized by causing phase separation of an antibiotic-containing solution of a mixed organic solvent that undergoes phase separation due to temperature changes.
目的とする抗生物質、たとえばマクロサイクリック・ラ
クタム・ラクトン系抗生物質であるミキソビレシンＡ
（Ｊ 、 Ｃｈｅｍ、Ｓｏｅ、、Ｃｈ＠ｍ、Ｃｏｍｍｕ
ｍ、 ＃１９８２．１３４０−１３４２（１９８２））
を任意の濃度含有した４５〜８０マ／マチアセトニトリ
ル均一水溶液を上部臨界完溶温＃（上部臨界共溶温度。Target antibiotic, such as myxoviresin A, a macrocyclic lactam lactone antibiotic
(J, Chem, Soe,, Ch@m, Commu
m, #1982.1340-1342 (1982))
A homogeneous aqueous solution of 45 to 80 mm/matiacetonitrile containing any concentration of
ＵＣ８Ｔなどともいう）以下で二相分離状態を維持する
温度範囲内、たとえは−５℃の雰囲気下で３時間以上攪
拌又は静置させる（なお、冷却時間はサンプル量により
上記時間以下でもよい。）。これＫよシ装置したサンプ
ルは、二相分離状態に変化する。攪拌したサンプルは、
静置させることにより懸濁状から二相に分離する。この
二相分離の上層は、アセトニトリルリッチな浴液でちゃ
、下層は水がリッチな液相もしくは固相となる。上層及
び下層のアセトニトリルｒａｗ＜モル分率）は、相分離
前のサングルの液組成と冷却温度によって定まる。こう
して、目的抗生物質たとえば、ミキソビレシンＡは上層
に選択的かつ定量的に抽出、回収される。また、上層の
液量については、相分離前のサンプルの液組成と冷却温
度によって定まるもので、任意に減少せしめることがで
きる。ここで、冷却温度は、サンプルの上部臨界完溶温
度以下であり、また、サンプルの完全凝固点（サンプル
がすべて凝固する温度）以上の温度であれば任意に設定
できるものである。このようにして、本発明は、目的抗
生物質を任意に濃縮することが可能であるばかりか、水
又は有機溶媒の除去も合わせて行うことができるという
特徴を有する。(also referred to as UC8T, etc.) within a temperature range that maintains a two-phase separated state, for example, in an atmosphere of -5°C, for 3 hours or more (note that the cooling time may be shorter than the above time depending on the amount of sample). ). A sample subjected to this K-type apparatus changes to a state of two-phase separation. The stirred sample is
By standing still, the suspension separates into two phases. The upper layer of this two-phase separation is an acetonitrile-rich bath liquid, and the lower layer is a water-rich liquid or solid phase. The acetonitrile raw<mole fraction) of the upper and lower layers is determined by the liquid composition of the sample before phase separation and the cooling temperature. In this way, the target antibiotic, for example myxoviresin A, is selectively and quantitatively extracted and recovered in the upper layer. Furthermore, the amount of liquid in the upper layer is determined by the liquid composition of the sample before phase separation and the cooling temperature, and can be reduced as desired. Here, the cooling temperature can be arbitrarily set as long as it is below the upper critical complete solution temperature of the sample and above the complete freezing point of the sample (the temperature at which all the sample solidifies). In this way, the present invention is characterized in that it is possible not only to arbitrarily concentrate the target antibiotic, but also to remove water or organic solvents.
また、目的抗生物質を含有する培養液にアセトニトリル
等の有機溶媒や酸・塩基・塩等を添加した系などで目的
抗生物質以外の沈殿が生じた場合は、遠心分離により沈
殿を除去し、上清を回収する。これを、至適の上部臨界
完１１１ｆ以下の温度に冷却させることによりニ相分離
させる。この時、培地由来成分や蛋白質成分等の不純物
と目的抗生物質を分離抽出させることができる。すなわ
ち、目的抗生物質の精製に相分離法を適用させることも
できる。このように、本発明の出発物質は目的抗生物質
を含有するものであれば特に限定されるものではない。In addition, if a precipitate other than the target antibiotic occurs in a system in which organic solvents such as acetonitrile, acids, bases, salts, etc. are added to the culture solution containing the target antibiotic, remove the precipitate by centrifugation, and then Collect the liquid. This is cooled to a temperature below the optimum upper critical temperature 111f to separate into two phases. At this time, impurities such as medium-derived components and protein components and the target antibiotic can be separated and extracted. That is, the phase separation method can also be applied to the purification of the target antibiotic. As described above, the starting material of the present invention is not particularly limited as long as it contains the objective antibiotic.
さらに、相分離法は、これを繰シ返すことにより、目的
抗生物質を徐々に濃縮させることも可能である。たとえ
は、前述のような一回相分離操作１１Ｊ７セトニトリル
のり、チな上層に水を更に添加する。その際、水の添加
量は、設定し次冷却温度での水のり、チな層の水濃度未
満の近傍とする。そして、相分離を行わしめ、目的抗生
物質を濃縮させる。以上の条件で相分離法を繰シ返すこ
とにより、目的抗生物質は、飽和濃度に近づく。Furthermore, by repeating the phase separation method, it is also possible to gradually concentrate the target antibiotic. For example, in a single phase separation operation as described above, water is further added to the top layer of the settonitrile paste. At this time, the amount of water added is set to be around less than the water concentration of the thick layer at the set next cooling temperature. Then, phase separation is performed to concentrate the target antibiotic. By repeating the phase separation method under the above conditions, the target antibiotic approaches a saturation concentration.
この方法全応用して、温和な条件で晶析を行うこともで
きる。その際、抗生物質の溶解度の下がる 。This method can also be applied to perform crystallization under mild conditions. At that time, the solubility of the antibiotic decreases.
操作をすればよく、二相分離状態から均一相へ変化させ
てもよいし、酸・塩基・塩類等を加えてもよい。Any operation may be performed, such as changing from a two-phase separated state to a homogeneous phase, or adding acids, bases, salts, etc.
以上、目的抗生物質が上層に選択的に濃縮・抽出・精製
される例をもって記述し念が、下層に目的抗生物質が濃
縮される場合についても、相分離の原理を適用すること
によって同様な操作をすることができる。The above description is based on an example in which the target antibiotic is selectively concentrated, extracted, and purified in the upper layer.However, similar operations can be performed by applying the principle of phase separation when the target antibiotic is concentrated in the lower layer. can do.
本発明で使用する有機溶媒溶液は、上記のアセトニトリ
ル−水系以外にその他のニトリル化合物−水系、酪酸−
水系、メチルグロピオン酸−水系等で、ｍｆ変化により
相分離を生ずる溶液系であれば制限されない。また、以
上の溶液の混合系であってもよい。In addition to the acetonitrile-water system mentioned above, the organic solvent solution used in the present invention may contain other nitrile compound-water systems, butyric acid-
There are no limitations as long as it is a solution system that causes phase separation due to a change in mf, such as an aqueous system or a methyl glopionic acid-aqueous system. Alternatively, a mixed system of the above solutions may be used.
さらに、上記溶液は、必要に応じて酸、塩基。Furthermore, the above solution may be mixed with an acid or a base as necessary.
塩−たとえば、トリフルオロ酢酸、ヘグタフルオロ酪酸
、リン酸、トリエチルアミンｐ Ｎａ２ＳＯ４ｐＮａＣ
ｌ 、 ＮａＮＯ３など一全含有する溶液系であっても
よい。Salts - for example trifluoroacetic acid, hegtafluorobutyric acid, phosphoric acid, triethylamine pNa2SO4pNaC
A solution system containing all of NaNO3 and NaNO3 may be used.
本発明で使用する抗生物質は、用いる有機溶媒系に安定
な抗生物質であれば制限されない。The antibiotic used in the present invention is not limited as long as it is stable in the organic solvent system used.
たトエば、ホスホマイシン、ストレプトマイシン、Ｄ−
シクロセリン、バシトラシン、アンピシリン、バンコマ
イシン、ブンブンビシン、ミキンビレシンＡ等の抗菌物
質、アドリアマイシン、ダウンマイシン、フスモマイシ
ンＡ、ストレグトスリシンＦ、マイトマイシンＣ，マイ
トマイシンＤ１等の制ガン物質、アンホテリシンＢ等の
抗真菌物質、イツリンＡ、パヒロマイシンＢ１等の微量
生理活性物質は当然のこと、それらを化学修飾した誘導
体にも適用できる。Tatoeba, Fosfomycin, Streptomycin, D-
Antibacterial substances such as cycloserine, bacitracin, ampicillin, vancomycin, bumbunbicin, mikinbirecin A, anticancer substances such as adriamycin, downmycin, fusmomycin A, stregtothricin F, mitomycin C, mitomycin D1, and antifungal substances such as amphotericin B , iturin A, pahilomycin B1, etc., as well as chemically modified derivatives thereof.
即ち、本発明は、抗生物質の種類及び由来等に制限され
ない極めて汎用性の高い濃縮精製法である。このように
本発明は極めて汎用性が高いわけであるが、その中でも
特に不安定な微量活性物質には有効である。そして、本
発明は操作、装置とも、非常に簡便であシ、スケ−ルア
ツブも極めて容易な方法である為１本発明を精製システ
ムに組み込むことで極めて効率的な精製を達成すること
が可能である。That is, the present invention is an extremely versatile concentration and purification method that is not limited by the type or origin of antibiotics. As described above, the present invention has extremely high versatility, and is particularly effective for unstable trace amounts of active substances. Furthermore, since the present invention is extremely simple in terms of operation and equipment, and is extremely easy to scale, it is possible to achieve extremely efficient purification by incorporating the present invention into a purification system. be.
なか、抗生物質以外の化合物、友とえば核酸。Among them, compounds other than antibiotics, such as nucleic acids.
アミノ酸、糖類等においても、有機溶媒に安定かつ相分
離法の適用が可能な物質であれば、本発明の方法を用い
て濃縮精製することができる。Amino acids, saccharides, etc. can also be concentrated and purified using the method of the present invention, as long as they are stable in organic solvents and can be subjected to phase separation.
以下、実施例により本発明の詳細な説明する。 Hereinafter, the present invention will be explained in detail with reference to Examples.
実施例１ ミキソビレシンＡ粗製溶液の濃縮精製ミキソ
ピレシンＡ７５ＩＲｇを含むミキソコッカス・フラベセ
ンスの菌体１００Ｌ（湿重量）にメタノール２１を加え
室温で２４時間抽出した。菌体を濾過で分離し、抽出液
１．９１（ミキソビレシンＡ７２．５■含有）を得た。Example 1 Concentration and Purification of Myxoviresin A Crude Solution To 100 L (wet weight) of Myxococcus flavescens cells containing Myxoviresin A75IRg was added 21 parts of methanol and extracted at room temperature for 24 hours. The bacterial cells were separated by filtration to obtain extract 1.91 (containing 72.5 μm of myxoviresin A).
とｎに水２．８５Ｊを加えて、ダイヤイオンＨＰ−２０
（三菱化成工業■製。Add 2.85 J of water to
(Made by Mitsubishi Chemical Industries, Ltd.)
０．５１）カラムに通液しミキソビレシンＡを吸着させ
た。６７％メタノール溶液１１．次に８０％メタノール
溶液１．５１で上記カラムを洗浄後１００％メタノール
０．５１でミキソピレシンＡを溶離した。溶離液（ミキ
ソビレシンＡ濃匣０．１３ダ／ａ）を減圧下濃縮乾固し
た。乾固物の総重量は４８０卿でミキソビレシンＡ６５
〜を含有した（純度１４％）。こｎを、本発明の原料と
する。0.51) Myxoviresin A was adsorbed by passing the solution through the column. 67% methanol solution 11. Next, the column was washed with 1.5 l of an 80% methanol solution, and Myxopyrescin A was eluted with 0.5 l of 100% methanol. The eluent (concentrated Myxoviresin A 0.13 Da/a) was concentrated to dryness under reduced pressure. The total weight of the dry matter was 480 kg, myxoviresin A65.
Contained ~ (purity 14%). This is used as the raw material of the present invention.
こ１料アセトニトリル５００−を加え溶解し次。これに
水５００１ｄｔ−７７０見１１とし７’ｃ（５０％アセ
トニトリル溶液）。遠心分離により不溶物を除去後９９
５ｄの遠心上滑液（ミキソビレシンＡ濃度０．０６４■
／−）を室温から一５℃に冷却させ３時間静置させた。Add and dissolve 500 ml of acetonitrile. To this, add water 5001dt-770 11 and 7'c (50% acetonitrile solution). After removing insoluble matter by centrifugation99
5 d of centrifuged synovial fluid (myxoviresin A concentration 0.064
/-) was cooled from room temperature to -5°C and allowed to stand for 3 hours.
こｎにより重液は二相分離をおこし次。その際、ミキソ
ビレシンＡは選択的に上層部（１４５ｍ／）に回収さｎ
、約５．５倍濃縮された（ミキソビレシンＡ濃度０．３
５■／ ｒｎｌ ）。そして、下層部には、黄色の着色
物質が抽出分離さｎた。This causes the heavy liquid to undergo two-phase separation. At that time, myxoviresin A was selectively collected in the upper layer (145 m/n).
, about 5.5 times concentrated (myxoviresin A concentration 0.3
5■/rnl). A yellow colored substance was extracted and separated from the lower layer.
上層部を減圧濃縮し、１５ｍの濃縮液を得た。The upper layer was concentrated under reduced pressure to obtain a 15 m concentrate.
こｎを遠心分離して沈殿画分を取シ乾燥し総重量１２６
ＷＩ９で４３■のミキソビレシンＡの含有物（純度３４
％）１−取得し次。This was centrifuged and the precipitate fraction was taken and dried to a total weight of 126
Myxoviresin A content of 43■ in WI9 (purity 34
%) 1-get next.
実施例２ ミキソビレシンＡ含有溶液の濃縮抽出実施例
１で得たミ゛キソビレシンＡ （ＷｔｊＸｆｉｋ １２
６■、ミキソビレシン人含有量４３■、純度３４％）を
５５％のアセトニトリル−水溶液４０ｄに溶解しＬＲＰ
−１カラム（ワットマン製品、２０１１１７）に通液し
吸着させた。次いで、６０％アセトニトリル溶液でミキ
ソピレシンＡを溶離した（純度９９．９％）。Example 2 Concentration and extraction of myxoviresin A-containing solution Myxoviresin A (WtjXfik 12) obtained in Example 1
6■, myxoviresin content 43■, purity 34%) was dissolved in 40d of 55% acetonitrile-aqueous solution and subjected to LRP.
-1 column (Whatman product, 201117) to adsorb the solution. Myxopyrescin A was then eluted with a 60% acetonitrile solution (99.9% purity).
溶離液３８ゴに水７．６１Ｌｔを加え５０％アセトニト
リル溶液とした（ミキソビレシンＡ２５．８１Ｒ９を有
）。7.61 Lt of water was added to the eluent 38 to make a 50% acetonitrile solution (containing myxoviresin A25.81R9).
こｎを２３℃から一５℃に温度変化させ３時間静置した
。均一相から二相に変化し表−１に示す結果が得られた
。The temperature was changed from 23°C to -5°C and the mixture was allowed to stand for 3 hours. The homogeneous phase changed to two phases, and the results shown in Table 1 were obtained.
すなわち、ミキソビレシンＡは上層部に８５％回収さｎ
、約５．６倍濃縮さｎた。That is, 85% of myxoviresin A was recovered in the upper layer.
, about 5.6 times concentrated.
表−１
実施例３ ダウノマイシン含有溶液の濃縮抽出放線菌の
生産する制ガン抗生物質（アンスラサイクリン系）であ
るダウノマイシン１１Ｎ？／ｍＡ溶液（７０％アセトニ
トリル−水）１０ｄを試験管に入ｎ２３℃から一５℃に
温度変化させ３時間静置させた。均一相から二相に変化
し、表−２に示す結果が得らｎた。すなわち、ダウノマ
イシンは下層部に８５％回収さｎ、約２．４倍濃縮され
た。Table 1 Example 3 Concentrated extraction of daunomycin-containing solution Daunomycin 11N, an anticancer antibiotic (anthracycline) produced by actinomycetes. 10 d of /mA solution (70% acetonitrile-water) was put into a test tube, the temperature was changed from 23°C to -5°C, and the mixture was allowed to stand for 3 hours. The homogeneous phase changed to two phases, and the results shown in Table 2 were obtained. That is, 85% of daunomycin was recovered in the lower layer, and it was concentrated about 2.4 times.
表−２
実施例４ パヒロマイシンＢ、含有溶液の濃縮抽出放線
菌の生産するＮａ”、Ｋ”−ＡＴＰａｓｓ阻害剤（マク
ロライド系抗生物質）パヒロマイシンＢ、 （Ｊ。Table 2 Example 4 Concentration extraction of pahilomycin B-containing solution Na'', K''-ATPass inhibitor (macrolide antibiotic) pahilomycin B (J.
Ａｎｔｉｂｉｏｔ、、 ３７．１１０−１１７（１９８
４） ） ０． Ｉ Ｗ／”溶液（５０％アセトニトリ
ル−水、ｐＨ６，９〜７．０）１０ｍ７を試験管に入ｎ
、２３℃から一５℃に温度変化させ３時間静置させた。Antibiot, 37.110-117 (198
4) ) 0. Pour 10 m7 of IW/” solution (50% acetonitrile-water, pH 6.9-7.0) into a test tube.
The temperature was changed from 23°C to -5°C, and the mixture was allowed to stand for 3 hours.
均一相から二相に変化し、表−３に示す結果が得らｎた
。The homogeneous phase changed to two phases, and the results shown in Table 3 were obtained.
すなわち、パヒロマイシンは上層に８５％回収さｎ約５
．７倍に濃縮された。また、本物質はメタノール存在の
酸性条件下にするとパヒロマイシンＢ２に変換し生物活
性が著しく低下するが、本発明の方法では、安定してパ
ヒロマイシンＢ、の濃縮精製できた。That is, pahilomycin is 85% recovered in the upper layer n about 5
．． Concentrated 7 times. Furthermore, when this substance is subjected to acidic conditions in the presence of methanol, it is converted to pahilomycin B2 and its biological activity is significantly reduced, but the method of the present invention allows stable concentration and purification of pahilomycin B.
表−３
〔発明の効果〕
本発明によｎば、目的抗生物質を極めて簡単に濃縮精製
することが可能である。こｎにより、粗製あるいは精製
工程での負荷が軽減されるだけでなく、液体クロマトグ
ラフィーの前処理あるいは後処理としても非常に有効で
ある。Table 3 [Effects of the Invention] According to the present invention, it is possible to concentrate and purify the target antibiotic extremely easily. This not only reduces the burden on crude or purification steps, but is also very effective as a pre-treatment or post-treatment for liquid chromatography.
Claims (1)
含有浴液を温度変化により相分離せしめることを特徴と
する抗生物質の濃縮精製方法。 ２、相分離した溶液を相互分離することを特徴とする請
求項１記載の抗生物質の濃縮精製方法。 ３、混合有機溶媒が酸、塩基又は塩類を含む請求項１記
載の抗生物質の濃縮精製方法。 ４、相分離の温度が上部臨界完溶温度以下で、完全凝固
点以上であることを特徴とする請求項１記載の抗生物質
の濃縮精製方法。 ５、抗生物質が微生物又は植物が産生する物質又は、そ
れを化学修飾した誘導体であることを特徴とする請求項
１記載の抗生物質の濃縮精製方法。[Scope of Claims] 1. A method for concentrating and purifying antibiotics, which comprises causing phase separation of an antibiotic-containing bath solution of a mixed organic solvent that undergoes phase separation due to temperature changes. 2. The method for concentrating and purifying antibiotics according to claim 1, which comprises mutually separating the phase-separated solutions. 3. The method for concentrating and purifying antibiotics according to claim 1, wherein the mixed organic solvent contains an acid, a base, or a salt. 4. The method for concentrating and purifying antibiotics according to claim 1, characterized in that the phase separation temperature is below the upper critical dissolution temperature and above the complete freezing point. 5. The method for concentrating and purifying antibiotics according to claim 1, wherein the antibiotic is a substance produced by a microorganism or a plant, or a chemically modified derivative thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1793888A JP2625809B2 (en) | 1988-01-28 | 1988-01-28 | Concentration and purification method of antibiotics |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1793888A JP2625809B2 (en) | 1988-01-28 | 1988-01-28 | Concentration and purification method of antibiotics |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01193280A true JPH01193280A (en) | 1989-08-03 |
| JP2625809B2 JP2625809B2 (en) | 1997-07-02 |
Family
ID=11957719
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1793888A Expired - Lifetime JP2625809B2 (en) | 1988-01-28 | 1988-01-28 | Concentration and purification method of antibiotics |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2625809B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003027049A1 (en) * | 2001-09-20 | 2003-04-03 | Ezaki Glico Co., Ltd. | Method of extracting and method of purifying an effective substance |
-
1988
- 1988-01-28 JP JP1793888A patent/JP2625809B2/en not_active Expired - Lifetime
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003027049A1 (en) * | 2001-09-20 | 2003-04-03 | Ezaki Glico Co., Ltd. | Method of extracting and method of purifying an effective substance |
| US7282150B2 (en) | 2001-09-20 | 2007-10-16 | Ezaki Glico Co., Ltd. | Method of extracting and method of purifying an effective substance |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2625809B2 (en) | 1997-07-02 |
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