JPH0196195A - Separation of bioprotein - Google Patents
Separation of bioproteinInfo
- Publication number
- JPH0196195A JPH0196195A JP62254875A JP25487587A JPH0196195A JP H0196195 A JPH0196195 A JP H0196195A JP 62254875 A JP62254875 A JP 62254875A JP 25487587 A JP25487587 A JP 25487587A JP H0196195 A JPH0196195 A JP H0196195A
- Authority
- JP
- Japan
- Prior art keywords
- glucomannan
- particles
- bioprotein
- liquid
- spherical particles
- 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.)
- Pending
Links
- 238000000926 separation method Methods 0.000 title abstract description 9
- 229920002581 Glucomannan Polymers 0.000 claims abstract description 28
- 229940046240 glucomannan Drugs 0.000 claims abstract description 28
- LUEWUZLMQUOBSB-FSKGGBMCSA-N (2s,3s,4s,5s,6r)-2-[(2r,3s,4r,5r,6s)-6-[(2r,3s,4r,5s,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5s,6r)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](OC3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-FSKGGBMCSA-N 0.000 claims abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 12
- 239000012798 spherical particle Substances 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 14
- 102000004169 proteins and genes Human genes 0.000 claims description 11
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 abstract description 18
- 239000012736 aqueous medium Substances 0.000 abstract description 14
- 239000002245 particle Substances 0.000 abstract description 12
- 239000002904 solvent Substances 0.000 abstract description 9
- 150000002148 esters Chemical class 0.000 abstract description 7
- 239000004372 Polyvinyl alcohol Substances 0.000 abstract description 4
- 229920002451 polyvinyl alcohol Polymers 0.000 abstract description 4
- 102000004506 Blood Proteins Human genes 0.000 abstract description 3
- 108010017384 Blood Proteins Proteins 0.000 abstract description 3
- 238000004132 cross linking Methods 0.000 abstract description 3
- 238000001704 evaporation Methods 0.000 abstract 1
- -1 glucomannan ester Chemical class 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 239000000243 solution Substances 0.000 description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 9
- 239000003431 cross linking reagent Substances 0.000 description 7
- 238000003756 stirring Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 239000007864 aqueous solution Substances 0.000 description 6
- 238000007127 saponification reaction Methods 0.000 description 6
- 239000011550 stock solution Substances 0.000 description 6
- 239000003085 diluting agent Substances 0.000 description 5
- 235000019441 ethanol Nutrition 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 4
- 102000006395 Globulins Human genes 0.000 description 4
- 108010044091 Globulins Proteins 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 238000009835 boiling Methods 0.000 description 4
- 229940025902 konjac mannan Drugs 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 102000009027 Albumins Human genes 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-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
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 229910052938 sodium sulfate Inorganic materials 0.000 description 3
- 235000011152 sodium sulphate Nutrition 0.000 description 3
- BBMCTIGTTCKYKF-UHFFFAOYSA-N 1-heptanol Chemical compound CCCCCCCO BBMCTIGTTCKYKF-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 229920000057 Mannan Polymers 0.000 description 2
- 102000004338 Transferrin Human genes 0.000 description 2
- 108090000901 Transferrin Proteins 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- 229920001222 biopolymer Polymers 0.000 description 2
- NNBZCPXTIHJBJL-UHFFFAOYSA-N decalin Chemical compound C1CCCC2CCCCC21 NNBZCPXTIHJBJL-UHFFFAOYSA-N 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 239000007863 gel particle Substances 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 2
- 239000012581 transferrin Substances 0.000 description 2
- KBPLFHHGFOOTCA-UHFFFAOYSA-N 1-Octanol Chemical compound CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 239000004971 Cross linker Substances 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 description 1
- 239000001856 Ethyl cellulose Substances 0.000 description 1
- ZZSNKZQZMQGXPY-UHFFFAOYSA-N Ethyl cellulose Chemical compound CCOCC1OC(OC)C(OCC)C(OCC)C1OC1C(O)C(O)C(OC)C(CO)O1 ZZSNKZQZMQGXPY-UHFFFAOYSA-N 0.000 description 1
- 102000008857 Ferritin Human genes 0.000 description 1
- 108050000784 Ferritin Proteins 0.000 description 1
- 238000008416 Ferritin Methods 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 239000005057 Hexamethylene diisocyanate Substances 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 102000003505 Myosin Human genes 0.000 description 1
- 108060008487 Myosin Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000005686 Serum Globulins Human genes 0.000 description 1
- 108010045362 Serum Globulins Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 102000009843 Thyroglobulin Human genes 0.000 description 1
- 108010034949 Thyroglobulin Proteins 0.000 description 1
- XSTXAVWGXDQKEL-UHFFFAOYSA-N Trichloroethylene Chemical group ClC=C(Cl)Cl XSTXAVWGXDQKEL-UHFFFAOYSA-N 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 150000008280 chlorinated hydrocarbons Chemical class 0.000 description 1
- XENVCRGQTABGKY-ZHACJKMWSA-N chlorohydrin Chemical compound CC#CC#CC#CC#C\C=C\C(Cl)CO XENVCRGQTABGKY-ZHACJKMWSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000019325 ethyl cellulose Nutrition 0.000 description 1
- 229920001249 ethyl cellulose Polymers 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 108010074605 gamma-Globulins Proteins 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- RRAMGCGOFNQTLD-UHFFFAOYSA-N hexamethylene diisocyanate Chemical compound O=C=NCCCCCCN=C=O RRAMGCGOFNQTLD-UHFFFAOYSA-N 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 239000003350 kerosene Substances 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 229960002175 thyroglobulin Drugs 0.000 description 1
- DVKJHBMWWAPEIU-UHFFFAOYSA-N toluene 2,4-diisocyanate Chemical compound CC1=CC=C(N=C=O)C=C1N=C=O DVKJHBMWWAPEIU-UHFFFAOYSA-N 0.000 description 1
- UBOXGVDOUJQMTN-UHFFFAOYSA-N trichloroethylene Natural products ClCC(Cl)Cl UBOXGVDOUJQMTN-UHFFFAOYSA-N 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Treatment Of Liquids With Adsorbents In General (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は生体蛋白質をクロマトグラフィ法によって分離
する方法に関するものである。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for separating biological proteins by chromatography.
ゲル濾過法は、分離技術として信頼性が高く、取り扱い
が簡単であることから酵素、多糖類。Gel filtration is a highly reliable separation technique and is easy to handle for enzymes and polysaccharides.
核酸、タンパク質などの分離精製の分野で重要な位置を
占めている。このゲル濾過法には充填剤として親水性ゲ
ル粒子が用いられる。例えばデキストラン、アガロース
、セルロース、ポリビニルアルコール及びポリアクリル
アミドなどの球状粒子が適用されている。It occupies an important position in the field of separation and purification of nucleic acids, proteins, etc. Hydrophilic gel particles are used as a filler in this gel filtration method. For example, spherical particles of dextran, agarose, cellulose, polyvinyl alcohol and polyacrylamide have been applied.
ところが、従来のゲル粒子では耐圧性、即ち強度が弱く
、高流速の条件で通液すると、運転中、通液圧力で粒子
が変形し、圧力損失が上昇し、分離効率が大巾に下がる
という問題があった。However, conventional gel particles have low pressure resistance, that is, strength, and when liquid is passed through at high flow rates, the particles are deformed by the pressure of liquid flow during operation, increasing pressure loss and significantly reducing separation efficiency. There was a problem.
そこで、本発明は、前記問題点を解決すると同時に、と
くに生体蛋白質の分離に有効な方法を開発することを目
的とするものである。Therefore, the present invention aims to solve the above-mentioned problems and at the same time to develop a method particularly effective for separating biological proteins.
とりわけ、血漿蛋白質であるアミラーゼ、トランスフェ
リン（β１−グロブリン）、γ−グロブリン、アルブミ
ン等の分離は血液の血漿成分分画製剤上極めて重要であ
り、これらは現在コーンのエタノール法（段階的にアル
コール濃度を変えて血漿蛋白質を分別沈澱する方法）、
アガロース系やデキストラン系ゲルを用いたカラムクロ
マトグラフィーにより分離されている。In particular, the separation of plasma proteins such as amylase, transferrin (β1-globulin), γ-globulin, and albumin is extremely important in preparing blood plasma component fractions, and these are currently being processed using Cohn's ethanol method (stepwise alcohol concentration). (method of fractional precipitation of plasma proteins by changing the
It is separated by column chromatography using agarose-based or dextran-based gels.
しかしコーン法は工程が多くて繁雑であり、又今のゲル
は流速がとれない（０，１〜０．３ｍＱ／ｍがせいぜい
）という問題があり、この解決が本発明の１つの目的で
ある。However, the cone method has many steps and is complicated, and current gels have the problem that the flow rate cannot be maintained (0.1 to 0.3 mQ/m at most), and one purpose of the present invention is to solve this problem. .
本発明は、架橋されたグルコマンナン球状粒子を充填し
たカラムに生体蛋白質を含む液を通液することを特徴と
する生体蛋白質の分離方法に関する。The present invention relates to a method for separating biological proteins, which comprises passing a liquid containing biological proteins through a column filled with cross-linked glucomannan spherical particles.
生体蛋白質としては、各種塩基、ヌクレオシド、ヌクレ
オチド、核酸、酵素、ホルモン、その他の蛋白質が含ま
れる。具体的には動物グロブリン、植物グロブリンを含
むグロブリン類、例えば；血清グロブリン、ミオシン、
インシュリン、ジデロフィリン（トランスフェリン）、
チログロブリン、リゾチーム；アルブミン例えば血清ア
ルブミン；アミラーゼ；フェリチン；チトクロムなどの
名前を挙げることができる。Biological proteins include various bases, nucleosides, nucleotides, nucleic acids, enzymes, hormones, and other proteins. Specifically, globulins including animal globulin and plant globulin, such as; serum globulin, myosin,
insulin, diderophylline (transferrin),
Names such as thyroglobulin, lysozyme; albumins such as serum albumin; amylase; ferritin; cytochromes may be mentioned.
本発明で使用される架橋されたグルコマンナン球状粒子
は、膨潤度１．５以上、排除限界分子量２００〜ｔｏｏ
、０００，０００（好ましくはｔｏ、ｏｏｏ以上）であ
って、かつ粒径１〜５００μｍのものである。The crosslinked glucomannan spherical particles used in the present invention have a swelling degree of 1.5 or more and an exclusion limit molecular weight of 200 to too
, 000,000 (preferably to, ooo or more) and a particle size of 1 to 500 μm.
架橋されたグルコマンナン球状粒子は、グルコマンナン
のエステルを単独に、又は希釈剤と共に、水性媒質より
沸点が低く、水性媒質に溶解しないか又は僅かしか溶解
しない溶媒中に溶解させた液を原液とし、この原液を水
性媒質中に懸濁させて液滴を診成させ、次いで液滴中の
溶媒を蒸発し、得られたグルコマンナンのエステルの球
状粒子をけん化し、架橋剤と反応させて架橋を行わせる
ことにより製造することができる。The crosslinked glucomannan spherical particles are prepared by dissolving glucomannan ester alone or together with a diluent in a solvent that has a boiling point lower than that of an aqueous medium and does not dissolve or only slightly dissolves in the aqueous medium. This stock solution is suspended in an aqueous medium to form droplets, then the solvent in the droplets is evaporated, the resulting spherical particles of glucomannan ester are saponified, and reacted with a crosslinking agent to form crosslinkers. It can be manufactured by performing the following steps.
本発明で用いるグルコマンナンのエステルは次のように
して製造する。The glucomannan ester used in the present invention is produced as follows.
即ち、市販のグルコマンナンをそのままか、又はグルコ
マンナンを一度水に溶解させ、次にエチルアルコール中
に沈澱させたコンニャクマンナン精製物をホルムアミド
又はジメチルホルムアミド等の溶媒に溶かし、触媒とし
てピリジンを用い、酸を加えてグルコマンナンのエステ
ルを作る。That is, commercially available glucomannan is used as it is, or glucomannan is dissolved in water and then precipitated in ethyl alcohol, and then purified konjac mannan is dissolved in a solvent such as formamide or dimethylformamide, using pyridine as a catalyst, Add acid to make glucomannan ester.
用いる酸としては酢酸、無水酢酸、プロピオン酸、醋酸
、硝酸など、任意の有機酸や無機酸が挙げられる。Examples of the acid used include any organic or inorganic acid such as acetic acid, acetic anhydride, propionic acid, acetic acid, and nitric acid.
酸は１種だけでなく、複数種用いて混合エステルを得て
もよい。A mixed ester may be obtained by using not only one type of acid but also multiple types of acids.
次に、こうして得られたエステルを溶媒に溶かすが、溶
媒としては、後記の水性媒質より沸点が低く、かつ水性
媒質に全く溶解しないか。Next, the ester thus obtained is dissolved in a solvent, but the solvent should have a boiling point lower than that of the aqueous medium described below, and be completely insoluble in the aqueous medium.
又は僅かしか溶解しないものであることが必要である。Or it needs to be something that only slightly dissolves.
具体的には、ジクロロメタン、クロロホルム、四塩化炭
素及びトリクロロエチレン等の塩素化炭化水素が使用さ
れ、これらを単独又は混合して用いる。Specifically, chlorinated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, and trichloroethylene are used, and these are used alone or in combination.
グルコマンナンのエステルの溶解濃度としては前記溶媒
が蒸発除去された後１粒子が球状を保ち、充填剤として
の強度を持っておればよいのであって１通常０．５〜２
０重量％、好ましくは２〜１０重量％とする。The dissolved concentration of glucomannan ester is usually 0.5 to 2, as long as each particle remains spherical after the solvent is evaporated and has the strength as a filler.
0% by weight, preferably 2 to 10% by weight.
なお、グルコマンナンのエステルを前記溶媒に溶かす際
、前記エステルを単独で溶かしてもよいが、適当な希釈
剤をさらに加えてもよい。Note that when dissolving the glucomannan ester in the solvent, the ester may be dissolved alone, or a suitable diluent may be further added.
希釈剤は球状粒子を作った後、除去されて球状粒子を多
孔化せしめるために使用されるもので、具体的には、テ
トラヒドロナフタレン、デカヒドロナフタレン、エチル
ベンゼン、ジエチルベンゼン、ドデカン酸メチル、トル
エン、ヘキシルアルコール、ヘプチルアルコール及びオ
クチルアルコール等が使われる。After the spherical particles are made, the diluent is removed and used to make the spherical particles porous.Specifically, the diluent is used to make the spherical particles porous. Alcohol, heptyl alcohol, octyl alcohol, etc. are used.
希釈剤の濃度としては使用するコンニャクマンナンのエ
ステルに対してｌＯ〜５００重景％、好ましくは５０〜
４００重量％である。The concentration of the diluent is 10 to 500%, preferably 50 to 50%, based on the konjac mannan ester used.
It is 400% by weight.
以上の操作の結果、グルコマンナンのエステルを含む原
液が得られるが、本発明では、次にこの原液を水性媒質
中に懸濁させる。As a result of the above operations, a stock solution containing glucomannan ester is obtained, and in the present invention, this stock solution is then suspended in an aqueous medium.
水性媒質としては親水性保護コロイド、例えばポリビニ
ルアルコール、カルボキシメチルセルロース、エチルセ
ルロース、メチルセルロース、可溶性澱粉並びにゼラチ
ン等が用いられる。As the aqueous medium, hydrophilic protective colloids such as polyvinyl alcohol, carboxymethyl cellulose, ethyl cellulose, methyl cellulose, soluble starch, and gelatin are used.
これらは０．１〜１０重量％、好ましくは１〜５重景％
水溶液として使用するのがよい。また、水性媒質の使度
量としてはコンニャクマンナンエステル溶液の少くとも
２倍以上好ましくは２〜５０倍容量とするのがよい。These are 0.1 to 10% by weight, preferably 1 to 5% by weight.
It is best used as an aqueous solution. The amount of the aqueous medium to be used is preferably at least twice the volume of the konjac mannan ester solution, preferably 2 to 50 times.
水性媒質中に前記原液を懸濁させる方法とし ・では、
水性媒質中に前記原液を全量加え、撹拌して分散、懸濁
する方法や、水性媒質を撹拌状態とし、これに前記原液
を一度に又は滴下状に添加する方法等が挙げられる。As a method of suspending the stock solution in an aqueous medium,
Examples include a method in which the entire amount of the stock solution is added to an aqueous medium and stirred to disperse or suspend, and a method in which the aqueous medium is stirred and the stock solution is added at once or dropwise to the aqueous medium.
液滴中の有機溶媒を蒸発除去する時の温度としては、水
性媒質の氷点以上で有機溶媒の沸点以下の温度が用いら
れるが、蒸発除去を促進させ、かつ粒子形状を良好に保
つためには有機溶媒の沸点より１〜５℃低い温度が好ま
しい。The temperature used to evaporate the organic solvent in the droplets is above the freezing point of the aqueous medium and below the boiling point of the organic solvent. The temperature is preferably 1 to 5°C lower than the boiling point of the organic solvent.
次にグルコマンナンのエステルの球状粒子をけん化する
が、その場合１球状粒子の形状をこわさずにその形状を
保ちつつ、けん化するようなけん化浴を用いることが必
要である。けん化浴の例としては水酸化ナトリウム又は
水酸化カリウムのメタノール溶液や、水酸化ナトリウム
又は水酸化カリウムを硫酸ナトリウム等の塩類水溶液に
溶解させた溶液が挙げられる。Next, the spherical particles of glucomannan ester are saponified, but in this case it is necessary to use a saponification bath that can saponify the spherical particles while maintaining their shape without destroying it. Examples of saponification baths include methanol solutions of sodium hydroxide or potassium hydroxide, and solutions in which sodium hydroxide or potassium hydroxide is dissolved in an aqueous salt solution such as sodium sulfate.
前者の例でけん化の具体的方法をのべると、グルコマン
ナンのエステルの球状粒子を予め５〜１０倍重量のメタ
ノールに１〜２時間浸漬したものに、マンナンエステル
の５０重量％に相当するメタノール（水酸化ナトリウム
水溶液含有）を添加し、室温で２４時間撹拌することに
よってけん化を行う。In the former example, the specific saponification method is as follows: Glucomannan ester spherical particles are immersed in 5 to 10 times the weight of methanol for 1 to 2 hours, and then methanol (corresponding to 50% by weight of the mannan ester) is added. Saponification is carried out by adding aqueous sodium hydroxide (containing an aqueous sodium hydroxide solution) and stirring at room temperature for 24 hours.
後者の例でけん化の具体的方法をのべるとグルコマンナ
ンのエステルの球状粒子をアルカリとして水酸化ナトリ
ウム又は水酸化カリウム。In the latter example, a specific method of saponification is to use spherical particles of glucomannan ester as an alkali and use sodium hydroxide or potassium hydroxide.
無機塩として硫酸ナトリウムを水に溶解した液に投入し
室温下２４時間撹拌を続けることによって行われる。This is carried out by adding sodium sulfate as an inorganic salt to a solution in water and continuing stirring at room temperature for 24 hours.
上記水溶液に対してアルカリ濃度は１０〜１５重景％で
あり、アルカリの量はグルコマンナンエステルに対して
５０重量％以上とする。硫酸ナトリウムは上記水溶液に
対して２０〜３０重量％とする。The alkali concentration in the aqueous solution is 10 to 15% by weight, and the amount of alkali is 50% by weight or more based on the glucomannan ester. The amount of sodium sulfate is 20 to 30% by weight based on the aqueous solution.
次にけん化されて得たグルコマンナンの粒子を架橋する
方法について述べる。Next, a method for crosslinking glucomannan particles obtained by saponification will be described.
架橋剤の例としてエピクロロヒドリン、ジェポキシブタ
ン、トリレンジイソシアナート、ヘキサメチレンジイソ
シアナート等の２官能性化合物を挙げることができる。Examples of crosslinking agents include difunctional compounds such as epichlorohydrin, jepoxybutane, tolylene diisocyanate, and hexamethylene diisocyanate.
これらの架橋剤は有機性媒体液中に溶解させて使用する
。These crosslinking agents are used after being dissolved in an organic medium.
架橋剤媒体液としては灯油又は流動パラフィン又はその
混合物（例えば容量比７：３）に界面活性剤（非イオン
界面活性剤例えばソルビタン脂肪酸エステル）を１〜２
重量％混合したものが用いられる。又別の架橋剤媒体液
としてはアセトンとジメチルスルホオキシドからなる混
合液（例えば容量比６：４）が用いられる。架橋剤の濃
度は上記架橋媒体液に対して０．Ｏ１〜１５ｍｏ Ｑ
／ Ｑの範囲である。The crosslinking agent medium liquid is kerosene or liquid paraffin or a mixture thereof (for example, volume ratio 7:3) and 1 to 2 surfactants (nonionic surfactants, for example, sorbitan fatty acid ester).
A mixture of % by weight is used. As another crosslinking agent medium solution, a mixed solution of acetone and dimethyl sulfoxide (eg, volume ratio 6:4) is used. The concentration of the crosslinking agent is 0. O1~15mo Q
/Q range.
架橋剤溶液１００容量部に対し、グルコマンナン球状粒
子を１〜５重量部加え、室温下に２４〜３６時間撹拌を
続けることによりグルコマンナンの球状粒子は架橋され
る。架橋反応粒子を決別し、アセトン次いで中性洗剤で
洗浄し次に水洗することによって架橋されたグルコマン
ナンの球状粒子が得られる。The glucomannan spherical particles are crosslinked by adding 1 to 5 parts by weight of glucomannan spherical particles to 100 volume parts of the crosslinking agent solution and continuing stirring at room temperature for 24 to 36 hours. Crosslinked glucomannan spherical particles are obtained by separating the crosslinked particles, washing with acetone, then with a neutral detergent, and then with water.
（１）架橋されたグルコマンナン球状粒子Ａ、Ｂの製造
コンニャクマンナン粉４０ｇを６０〜７０℃のイオン交
換水４Ｑに加え撹拌溶解した。これを６Ｑのエタノール
中に少しづつ滴下し、沈澱させ、決別、風乾１．２０℃
で真空乾燥させた。(1) Production of crosslinked glucomannan spherical particles A and B 40 g of konjac mannan powder was added to 4Q of ion-exchanged water at 60 to 70°C and dissolved with stirring. This was dropped little by little into 6Q ethanol, precipitated, separated, and air-dried at 1.20°C.
It was dried in vacuum.
この乾燥マンナン３０ｇをホルムアミドＩＱ中に入れ、
２日間膨潤させた後、ピリジン３００ＩＩＩＱ、無水酢
酸３００ｍ Ｑを加え、５０℃で５日間反応させた。こ
の反応混合物を７Ｑの水中に撹拌しながら加えた。生じ
た沈澱を決別、更に水洗した。これを風乾、真空乾燥し
た後、その３ｇをデカヒドロナフタリン７ｍＱ（ゲルＡ
）又は１０．５ｍＱ（ゲルＢ）を含むクロロホルム３０
０ｍＱに溶解した。、５７℃の９０％ケン化ポリビニル
アルコールの２％水溶液３Ｑ中に撹拌しながら上記溶液
を滴下し、撹拌して、粒径４４〜１０５μｍの粒子を形
成させた。２４時間後徐冷し、球状粒子を炉別し、水洗
した。これをメタノール２７０ｍ Ｑ、ｌ０Ｎ−ＮａＯ
Ｈ液３０ｍ Ｑ中に加え、撹拌下、２４時間放置し、ケ
ン化した。その後ケン化球状粒子を決別し、アセトンと
ジメチルスルホオキシド１：１の混合溶液３００ｍｎ中
にエビクロロヒドリン３５ｇを加えた架橋浴中に球状粒
子を投入し、６０℃で２４時間処理した。Put 30g of this dry mannan into formamide IQ,
After swelling for 2 days, pyridine 300IIIQ and acetic anhydride 300mQ were added, and the mixture was reacted at 50°C for 5 days. The reaction mixture was added to 7Q water with stirring. The resulting precipitate was separated and further washed with water. After air-drying and vacuum-drying this, 3 g of it was added to 7 mQ of decahydronaphthalene (gel A).
) or chloroform 30 containing 10.5 mQ (gel B)
Dissolved in 0mQ. The above solution was dropped into a 2% aqueous solution 3Q of 90% saponified polyvinyl alcohol at 57° C. with stirring, and the solution was stirred to form particles with a particle size of 44 to 105 μm. After 24 hours, the mixture was slowly cooled, and the spherical particles were separated in a furnace and washed with water. This was mixed with methanol 270m Q, 10N-NaO
The mixture was added to 30 m of H solution and left to stand for 24 hours with stirring to saponify it. Thereafter, the saponified spherical particles were separated, and the spherical particles were placed in a crosslinking bath in which 35 g of shrimp chlorohydrin was added to 300 mL of a mixed solution of acetone and dimethyl sulfoxide 1:1, and treated at 60° C. for 24 hours.
（２）各種生体蛋白質の分離
上記製造法で得られたグルコマンナン球状粒子Ａ、Ｂを
内径６ｍｍのステンレスカラムに充填高さが２５０ｍｍ
となるようにそれぞれ充填した。各種生体高分子物質を
１ ｍｇ／ｍ Ｑ含む０、ＩＭ Ｎａ２ＨＰＯ，、０，
３Ｍ ＮａＣＱ水溶液をｌμｆｌ注入し、０．ＩＭ Ｎ
ａ２’ＨＰＯ，、０，３Ｍ ＮａＣＱ水溶液を１．０ｍ
Ｑ ／ｍｉｎの流速で通液して溶出させた。各物質の
溶出容量は、Ｕｖ検出器を用いて測定した。得られた結
果からカラム容量（Ｖｔ）に対する各物質の溶出容量（
Ｖｅ）の比： Ｖｅ／Ｖｔ（％）の値を求めた。(2) Separation of various biological proteins Glucomannan spherical particles A and B obtained by the above production method were packed into a stainless steel column with an inner diameter of 6 mm and a height of 250 mm.
Each was filled so that 0, IM Na2HPO,,0, containing various biopolymer substances at 1 mg/m Q
1μfl of 3M NaCQ aqueous solution was injected, and 0. IM N
a2'HPO, 1.0 m of 0.3M NaCQ aqueous solution
Elution was carried out by passing the solution at a flow rate of Q/min. The elution volume of each substance was measured using a UV detector. From the obtained results, the elution capacity of each substance (
Ratio of Ve): The value of Ve/Vt (%) was determined.
結果を第１表に示す。各、生体高分子物質は分子量の大
きさの順に溶出してくることを確認できた。
・
第１表
このデータから、特に血漿分画製剤であるグロブリン、
アルブミンなどが容易に分取できることがわかる。The results are shown in Table 1. It was confirmed that each biopolymer substance was eluted in order of molecular weight.
・ From this data in Table 1, it is clear that globulin, which is a plasma-derived preparation,
It can be seen that albumin and the like can be easily separated.
本発明によれば、生体蛋白質を含む液を従来のゲル粒子
充填カラムでは通液できないような高流速の条件で通液
して分離できるため、分離効率が大巾に上昇する。又、
本発明で用いる架橋されたグルコマンナン球状粒子は高
強度なので層高を高くすることができるため分前性能が
大巾に向上する。According to the present invention, it is possible to separate a liquid containing biological proteins by passing the liquid through the column at a high flow rate that cannot be passed through a conventional gel particle-packed column, thereby greatly increasing the separation efficiency. or,
Since the crosslinked glucomannan spherical particles used in the present invention have high strength, the layer height can be increased, and the dispensing performance is greatly improved.
Claims (1)
ムに、生体蛋白質を含む液を通液することを特徴とする
生体蛋白質の分離方法。1. A method for separating biological proteins, which comprises passing a liquid containing biological proteins through a column filled with cross-linked glucomannan spherical particles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62254875A JPH0196195A (en) | 1987-10-09 | 1987-10-09 | Separation of bioprotein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62254875A JPH0196195A (en) | 1987-10-09 | 1987-10-09 | Separation of bioprotein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0196195A true JPH0196195A (en) | 1989-04-14 |
Family
ID=17271046
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62254875A Pending JPH0196195A (en) | 1987-10-09 | 1987-10-09 | Separation of bioprotein |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0196195A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5034466A (en) * | 1990-04-30 | 1991-07-23 | Shell Oil Company | Polymer and process for making the same |
| US5055536A (en) * | 1990-08-09 | 1991-10-08 | Shell Oil Company | Process to prepare vinyl ether polymers |
| US5068294A (en) * | 1990-09-28 | 1991-11-26 | Shell Oil Company | Process to produce polymers of styrene derivatives |
| US5073610A (en) * | 1990-09-28 | 1991-12-17 | Shell Oil Company | Polymers of alkoxystyrenes and a process to produce polymers of alkoxystyrenes |
-
1987
- 1987-10-09 JP JP62254875A patent/JPH0196195A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5034466A (en) * | 1990-04-30 | 1991-07-23 | Shell Oil Company | Polymer and process for making the same |
| US5055536A (en) * | 1990-08-09 | 1991-10-08 | Shell Oil Company | Process to prepare vinyl ether polymers |
| US5068294A (en) * | 1990-09-28 | 1991-11-26 | Shell Oil Company | Process to produce polymers of styrene derivatives |
| US5073610A (en) * | 1990-09-28 | 1991-12-17 | Shell Oil Company | Polymers of alkoxystyrenes and a process to produce polymers of alkoxystyrenes |
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