JPH0211601B2 - - Google Patents
Info
- Publication number
- JPH0211601B2 JPH0211601B2 JP62073439A JP7343987A JPH0211601B2 JP H0211601 B2 JPH0211601 B2 JP H0211601B2 JP 62073439 A JP62073439 A JP 62073439A JP 7343987 A JP7343987 A JP 7343987A JP H0211601 B2 JPH0211601 B2 JP H0211601B2
- Authority
- JP
- Japan
- Prior art keywords
- granular porous
- chitosan
- chitin
- solution
- activated
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 229920002101 Chitin Polymers 0.000 claims description 24
- 229920001661 Chitosan Polymers 0.000 claims description 21
- -1 cyanogen halide Chemical class 0.000 claims description 13
- JMANVNJQNLATNU-UHFFFAOYSA-N glycolonitrile Natural products N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 claims description 12
- 238000004519 manufacturing process Methods 0.000 claims 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 19
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 239000007864 aqueous solution Substances 0.000 description 9
- 239000008187 granular material Substances 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 239000002245 particle Substances 0.000 description 8
- 235000011121 sodium hydroxide Nutrition 0.000 description 8
- 229920000936 Agarose Polymers 0.000 description 6
- 108010074605 gamma-Globulins Proteins 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 230000007935 neutral effect Effects 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 4
- 230000002378 acidificating effect Effects 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 3
- 239000003637 basic solution Substances 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102000001554 Hemoglobins Human genes 0.000 description 2
- 108010054147 Hemoglobins Proteins 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 239000003929 acidic solution Substances 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 239000012670 alkaline solution Substances 0.000 description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 2
- 239000004327 boric acid Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000015271 coagulation Effects 0.000 description 2
- 238000005345 coagulation Methods 0.000 description 2
- 230000006196 deacetylation Effects 0.000 description 2
- 238000003381 deacetylation reaction Methods 0.000 description 2
- JXTHNDFMNIQAHM-UHFFFAOYSA-N dichloroacetic acid Chemical compound OC(=O)C(Cl)Cl JXTHNDFMNIQAHM-UHFFFAOYSA-N 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 238000004438 BET method Methods 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 239000012345 acetylating agent Substances 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 125000003158 alcohol group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- QPJDMGCKMHUXFD-UHFFFAOYSA-N cyanogen chloride Chemical compound ClC#N QPJDMGCKMHUXFD-UHFFFAOYSA-N 0.000 description 1
- 238000007872 degassing Methods 0.000 description 1
- 229960005215 dichloroacetic acid Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 229960001922 sodium perborate Drugs 0.000 description 1
- YKLJGMBLPUQQOI-UHFFFAOYSA-M sodium;oxidooxy(oxo)borane Chemical compound [Na+].[O-]OB=O YKLJGMBLPUQQOI-UHFFFAOYSA-M 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明はアミノ基を有する、例えば、蛋白質、
ペプチド、アミノ酸等を容易に固定化出来る新規
なハロゲン化シアン活性化粒状多孔質キチンに関
し、このものは、アフイニテイクロマトグラフイ
ー用担体、酵素固定化用担体等の分野に好適なも
のである。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to proteins having an amino group, such as proteins,
The present invention relates to a novel halogenated cyanide-activated granular porous chitin that can easily immobilize peptides, amino acids, etc., and is suitable for use in fields such as affinity chromatography carriers and enzyme immobilization carriers.
従来、特公昭57―21529号に開示されている如
く、臭化シアンによつて活性化されたアガロース
は知られており、ハロゲン化シアンで活性化すれ
ば分子中の水酸基と反応しアミノ基と反応し易く
なる。しかし、上記公報に開示されているアガロ
ースやデキストランの如き多糖類等のゲルをハロ
ゲン化シアンで活性化したものをアフイニテイー
クロマトグラフイーや固定化酵素用担体に供した
場合には、該多糖類はゲル状であつて、強度が不
足するため通液時の圧力上昇が著しくなり、実用
的でない欠点がある。
Conventionally, as disclosed in Japanese Patent Publication No. 57-21529, agarose activated with cyanogen bromide has been known, and when activated with cyanogen halide, it reacts with the hydroxyl group in the molecule and forms an amino group. It becomes easier to react. However, when the gel of polysaccharides such as agarose and dextran disclosed in the above publication is activated with cyanogen halide and used for affinity chromatography or as a carrier for immobilized enzymes, the polysaccharides is in the form of a gel and lacks strength, resulting in a significant pressure rise during liquid passage, which is impractical.
本発明は上述の如く、工業的に使用する場合
に、圧損を生じない様な強度を有する蛋白質、ペ
プチドアミノ酸等を容易に固定化できる新規なハ
ロゲン化シアン活性化多糖類を得ることを目的と
し、本発明者等の開発した粒状多孔質キチンを用
いることによつて上記問題点を解決した。
As mentioned above, the purpose of the present invention is to obtain a novel cyanogen halide-activated polysaccharide that can easily immobilize proteins, peptide amino acids, etc., and has a strength that does not cause pressure loss when used industrially. The above problems were solved by using granular porous chitin developed by the present inventors.
本発明は、粒状多孔質キトサンを無水酢酸等の
アセチル化剤でＮ―アセチル化して粒状多孔質再
生キチンとした後に、アルカリ条件下でハロゲン
化シアンと反応させて得られる、ハロゲン化シア
ン活性化粒状多孔質キチンに関するものである。
The present invention provides activated cyanogen halide, which is obtained by N-acetylating granular porous chitosan with an acetylating agent such as acetic anhydride to obtain granular porous regenerated chitin, and then reacting it with cyanogen halide under alkaline conditions. It concerns granular porous chitin.
粒状多孔質キトサンは、低分子量キトサンの酸
性溶液を塩基性溶液中に落下させて得られる。本
発明に用いる低分子量キトサンは平均分子量が
10000〜230000のものである。 Granular porous chitosan is obtained by dropping an acidic solution of low molecular weight chitosan into a basic solution. The low molecular weight chitosan used in the present invention has an average molecular weight of
10,000 to 230,000.
低分子量キトサンは、フレーク状の高分子量キ
トサンを、過硼酸ソーダ水溶液中で加温処理する
ことにより、所望の低い分子量を有する良質なキ
トサンが得られる。 High-quality chitosan having a desired low molecular weight can be obtained by heating flaky high-molecular-weight chitosan in an aqueous sodium perborate solution.
低分子量キトサンは、酢酸、ジクロル酢酸、蟻
酸の単独又は混合物の水溶液に溶解させてキトサ
ン酸性溶液とする。その濃度は取扱いの容易な範
囲を適宜選択出来るが、２〜20％の範囲が好まし
い。 Low molecular weight chitosan is dissolved in an aqueous solution of acetic acid, dichloroacetic acid, and formic acid alone or as a mixture to obtain an acidic chitosan solution. The concentration can be appropriately selected within a range that is easy to handle, but a range of 2 to 20% is preferred.
該キトサン酸性溶液を孔径0.1〜0.25mmφのノ
ズルより圧力下で塩基性凝固液中に一定量ずつ落
下させることによつて粒状多孔質キトサンが得ら
れる。 Granular porous chitosan is obtained by dropping a fixed amount of the chitosan acidic solution into the basic coagulation liquid under pressure through a nozzle with a pore size of 0.1 to 0.25 mmφ.
凝固浴の塩基性物質としては、水酸化ナトリウ
ム、水酸化カリウム、炭酸ナトリウム、炭酸カリ
ウム、アンモニア、エチレンジアミン等のアルカ
リ性物質が用いられ、塩基性物質には、水又はメ
タノール、エタノール等の極性を有するアルコー
ル類又は水とアルコールとの混合物に前記塩基性
物質を加えて使用する。得られた粒状多孔質キト
サンは極性溶媒を用いて中性になる迄充分洗浄を
行う。この様にして得られた粒状多孔質キトサン
をアルコールで水置換後、例えばエタノール中で
無水酢酸を用いて反応させ、Ｎ―アセチル化を行
い、更に苛性ソーダ水溶液の如きアルカリ性溶液
で処理してエステル結合を切断して完全なキチン
化を計り、粒状多孔質再生キチンとする。 As basic substances in the coagulation bath, alkaline substances such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, ammonia, and ethylenediamine are used, and basic substances include water or polar substances such as methanol and ethanol. The basic substance is added to alcohol or a mixture of water and alcohol. The obtained granular porous chitosan is thoroughly washed with a polar solvent until it becomes neutral. After replacing the water with alcohol, the granular porous chitosan thus obtained is reacted with acetic anhydride in ethanol to perform N-acetylation, and further treated with an alkaline solution such as an aqueous solution of caustic soda to form an ester bond. is cut to ensure complete chitinization, resulting in granular porous regenerated chitin.
該粒状多孔質再生キチンを苛性ソーダ水溶液の
如きアルカリ性溶液でPHを10〜20に調節しながら
ハロゲン化シアンを反応させる。ハロゲン化シア
ンとしては、臭化シアン、塩化シアンが使用され
る。かくして得られたハロゲン化シアン活性化粒
状多孔質キチンはキチン質から成つているので結
晶構造にも優れ、強度に富んだ粒状物質で、粒状
物の表面及び割断面を観察してわかるように均質
に形成された微細孔を有する多孔性粒状物であ
る。従つて、表面積も大きいのでハロゲン化シア
ンの結合量も多くなり、活性化能も増大し、アミ
ノ基を有する蛋白質、ペプチド、アミノ酸等の固
定化能も極めて大きい特色がある。 The granular porous regenerated chitin is reacted with cyanogen halide while adjusting the pH to 10 to 20 with an alkaline solution such as an aqueous solution of caustic soda. As the cyanogen halide, cyanogen bromide and cyanogen chloride are used. The cyanogen halide activated granular porous chitin thus obtained is composed of chitin, so it has an excellent crystalline structure, is a strong granular material, and is homogeneous as can be seen by observing the surface and cut surface of the granular material. It is a porous granule having micropores formed in it. Therefore, since the surface area is large, the amount of cyanogen halide bound is increased, the activation ability is also increased, and the ability to immobilize proteins, peptides, amino acids, etc. having amino groups is also extremely high.
以下、実施例について本発明を説明するが、本
発明はこの範囲に限定されるものではない。 The present invention will be described below with reference to Examples, but the present invention is not limited to this scope.
実施例において比表面積は、試料を液体窒素中
で急冷凍結し、10-4トール、−40℃、８時間真空
乾燥し、140℃、40分間脱ガス後、比表面積自動
測定装置（島津マイクロメリテイツクス2200型）
にてBET法で測定した。 In the examples, the specific surface area was determined by rapidly freezing the sample in liquid nitrogen, vacuum drying at 10 -4 Torr, -40°C for 8 hours, degassing at 140°C for 40 minutes, and then using an automatic specific surface area measuring device (Shimadzu Micromeri). Teitskus 2200 type)
Measured using the BET method.
実施例 １
脱アセチル化度82％、平均分子量48000のキト
サン60ｇを酢酸30ｇを含む水940ｇ中に溶解して
固形分濃度６重量％のキトサン酸性溶液を得た。
該キトサン酸性溶液を７％苛性ソーダ、30％メタ
ノール、63％水からなる塩基性溶液中に孔径0.15
mmφのノズルより噴霧落下させて、これを中性に
なる迄水洗し、平均粒径0.1mmφ、比表面積64.0
m2／ｇの粒状多孔質キトサン１を得た。Example 1 60 g of chitosan having a degree of deacetylation of 82% and an average molecular weight of 48,000 was dissolved in 940 g of water containing 30 g of acetic acid to obtain an acidic chitosan solution having a solid content concentration of 6% by weight.
The acidic chitosan solution was placed in a basic solution consisting of 7% caustic soda, 30% methanol, and 63% water with a pore size of 0.15.
The spray is dropped from a mmφ nozzle and washed with water until it becomes neutral, with an average particle diameter of 0.1 mmφ and a specific surface area of 64.0.
Granular porous chitosan 1 of m 2 /g was obtained.
この50mlを採取し、エタノールで４回置換し、
水を完全にアルコールで置換した。このものに３
倍モルの無水酢酸を加え、エタノール中で24時間
撹拌し、Ｎ―アセチル化させ中性になる迄洗浄
し、更に、1N―NaOH50mlを加え１時間処理し、
中性になる迄充分水洗した。平均粒径0.1mmφ、
比表面積64.0m2／ｇの粒状多孔質キチン50mlが得
られた。 Collect 50ml of this, replace it with ethanol 4 times,
Water was completely replaced with alcohol. 3 for this thing
Add twice the mole of acetic anhydride, stir in ethanol for 24 hours, wash until N-acetylated and neutral, then add 50 ml of 1N-NaOH and treat for 1 hour.
It was thoroughly washed with water until it became neutral. Average particle size 0.1mmφ,
50 ml of granular porous chitin with a specific surface area of 64.0 m 2 /g was obtained.
更に、該粒状多孔質キチン50mlを250mlの水に
懸濁し、5N―苛性ソーダでPHを10〜12とした。
25ｇ／250mlの臭化シアン水溶液を少量ずつ撹拌
下に、PHを10〜12に制御しながら5N―苛性ソー
ダを加え反応させる。250ml臭化シアン水溶液の
添加が終了した後、更に30分間撹拌し、冷水で良
く洗浄して臭化シアン活性化粒状多孔質キチン44
mlを得た。 Further, 50 ml of the granular porous chitin was suspended in 250 ml of water, and the pH was adjusted to 10 to 12 with 5N caustic soda.
25g/250ml of cyanogen bromide aqueous solution is stirred little by little, and 5N caustic soda is added while controlling the pH to 10-12 for reaction. After the addition of 250 ml of cyanogen bromide aqueous solution is completed, stir for another 30 minutes and wash well with cold water to prepare cyanogen bromide activated granular porous chitin 44.
Got ml.
上記のようにして得られた平均粒径0.1mmφの
臭化シアン活性化粒状多孔質キチンと粒径が0.1
mmφのアガロースの臭化シアン活性化粒状物（フ
アルマシアフアインケミカル製、CNBrセフアロ
ース）を梅谷精機製の長さ25cm、径0.8cmの耐圧
カラム（150Kg／cm2）を用いて充填し、水を一定
流量流した時の圧力上昇度合を調べた結果、図面
に示す如くであつた。本発明の臭化シアン活性化
粒状多孔質キチンは強度に優れているため、通液
による圧力の上昇が小さく、アガロース粒状物に
比べ優れていることが判る。 Cyanogen bromide activated granular porous chitin with an average particle size of 0.1 mmφ obtained as above and a particle size of 0.1 mmφ
Cyanogen bromide-activated granules of agarose of mmφ (manufactured by Pharmacia Huain Chemical, CNBr Sepharose) were packed using a pressure-resistant column (150 Kg/cm 2 ) with a length of 25 cm and a diameter of 0.8 cm manufactured by Umetani Seiki. The results of examining the degree of pressure rise when water was flowed at a constant flow rate were as shown in the drawing. It can be seen that the cyanogen bromide activated granular porous chitin of the present invention has excellent strength, so that the increase in pressure due to liquid passage is small, and is superior to agarose granules.
更に、得られた臭化シアン活性化粒状多孔質キ
チン１mlを採取し、PH９，0.1M硼酸緩衝溶液で
洗浄した後、２mlの１％γ―グロブリン溶液を加
え、２時間振盪してγ―グロブリンを固定化し
た。固定化量を残液の吸光度と原液の吸光度の差
から求めた処、5.46mg／mlであつた。 Furthermore, 1 ml of the resulting cyanogen bromide-activated granular porous chitin was collected, washed with PH9, 0.1M boric acid buffer solution, 2 ml of 1% γ-globulin solution was added, and the mixture was shaken for 2 hours to remove γ-globulin. was fixed. The amount of immobilization was determined from the difference between the absorbance of the residual solution and the absorbance of the stock solution, and was 5.46 mg/ml.
また、比較のため、粒径が0.1mmφの上記のア
ガロース臭化シアン活性化粒状物１mlを用いて同
様の条件でγ―グロブリンを固定化し、固定化量
を残液の吸光度と原液の吸光度の差から求めたと
ころ、3.7mg／mlであつた。 For comparison, γ-globulin was immobilized under the same conditions using 1 ml of the above agarose cyanogen bromide activated granules with a particle size of 0.1 mmφ, and the amount of immobilization was determined by the absorbance of the remaining solution and the absorbance of the stock solution. When calculated from the difference, it was 3.7 mg/ml.
実施例 ２
実施例１で得た粒状多孔質キチンに１％γ―グ
ロブリンの代りに１％ヘモグロビンを用いて実施
例１と同量、同操作により固定したところ、ヘモ
グロビンの固定化量は3.05mg／mlであつた。Example 2 When the granular porous chitin obtained in Example 1 was immobilized using the same amount and procedure as in Example 1 using 1% hemoglobin instead of 1% γ-globulin, the amount of hemoglobin immobilized was 3.05 mg. /ml.
実施例 ３
脱アセチル化度78％、平均分子量65000のキト
サン70ｇを酢酸35ｇを含む水930ｇ中に溶解し、
固形分濃度７％のキトサン酸性水溶液を得た。該
キトサン酢酸溶液を10％苛性ソーダ、30％メタノ
ール、60％水からなる塩基性溶液中に、孔径0.25
mmφのノズルより落下させてこれを中性になる迄
水洗し、平均粒径1.0mmφ、比表面積85m2／ｇの
粒状多孔質キトサン１を得た。この50mlを採取
しエタノールで４回洗浄し、水を置換した。そし
て３倍モルの無水酢酸を加え、エタノール中で24
時間撹拌し中性になる迄水洗し、更に1N―苛性
ソーダ50mlを加えた後完全に水洗し、平均粒径
1.0mmφ、比表面積75m2／ｇの粒状多孔質キチン
50mlを得た。Example 3 70 g of chitosan with a degree of deacetylation of 78% and an average molecular weight of 65,000 was dissolved in 930 g of water containing 35 g of acetic acid,
A chitosan acidic aqueous solution with a solid content concentration of 7% was obtained. The chitosan acetic acid solution was placed in a basic solution consisting of 10% caustic soda, 30% methanol, and 60% water with a pore size of 0.25.
It was dropped through a mmφ nozzle and washed with water until it became neutral, yielding granular porous chitosan 1 having an average particle diameter of 1.0 mmφ and a specific surface area of 85 m 2 /g. 50 ml of this was collected and washed four times with ethanol, and the water was replaced. Then, 3 times the mole of acetic anhydride was added, and 24
Stir for hours, wash with water until neutral, then add 50ml of 1N caustic soda, wash completely with water, average particle size
Granular porous chitin with a diameter of 1.0 mm and a specific surface area of 75 m 2 /g.
Obtained 50ml.
該粒状多孔質キチン50mlを250mlの水に懸濁し、
5N―苛性ソーダでPH10〜12に制御した。25ｇ／
250mlの臭化シアン水溶液を少量ずつPHを10〜12
に5N―苛性ソーダで調節しながら加え反応させ
る。 Suspend 50ml of the granular porous chitin in 250ml of water,
The pH was controlled to 10-12 with 5N-caustic soda. 25g/
Add 250ml of cyanogen bromide aqueous solution in small portions to bring the pH to 10-12.
Add 5N-caustic soda while adjusting and react.
250mlの臭化シアン水溶液を添加終了後更に30
分撹拌し、冷水でよく洗浄して臭化シアン活性化
粒状多孔質キチン44mlを得た。 After adding 250ml of cyanogen bromide aqueous solution, add another 30ml of cyanogen bromide aqueous solution.
After stirring for several minutes and thoroughly washing with cold water, 44 ml of cyanogen bromide activated granular porous chitin was obtained.
上記のようにして得られた臭化シアン活性化粒
状多孔質キチン１mlを採取し、PH９，0.1M硼酸
緩衝溶液で軽く洗浄した後、２mlの１％γ―グロ
ブリン溶液を加え、２時間振盪してγ―グロブリ
ンを固定した。固定化量は、3.74mg／mlであつ
た。 1 ml of the cyanogen bromide-activated granular porous chitin obtained as above was collected, washed lightly with PH9, 0.1M boric acid buffer solution, 2 ml of 1% γ-globulin solution was added, and the mixture was shaken for 2 hours. γ-globulin was immobilized. The immobilized amount was 3.74 mg/ml.
本発明によるハロゲン化シアン活性化粒状多孔
質キチンは、充分な強度を有する多孔質の粒状体
であつて、上記実施例１及び図面に記載のように
カラム充填剤として用いても充分な強度を有する
ため通液時に圧損を生じず、工業的な利用に極め
て適したものである。
The cyanogen halide activated granular porous chitin according to the present invention is a porous granular material having sufficient strength, and has sufficient strength even when used as a column packing material as described in Example 1 and the drawings above. Because of this, no pressure loss occurs during liquid passage, making it extremely suitable for industrial use.
また、本発明のハロゲン化シアン活性化粒状多
孔質キチンは、粒径の揃つた、粒状体の表面及び
内部に均質に形成された微細孔を有し、比表面積
の大きい粒状体であるため、蛋白質やペプチドア
ミノ酸等を多量に固定化することができる。 In addition, the cyanogen halide-activated granular porous chitin of the present invention is a granular material with uniform particle sizes, homogeneously formed on the surface and inside of the granular material, and has a large specific surface area. Large amounts of proteins, peptide amino acids, etc. can be immobilized.
図面は、本発明による臭化シアン活性化粒状多
孔質キチンと、従来のアガロースの臭化シアン活
性化粒状物とをカラムに充填した際の、通液時の
圧力上昇を示すグラフである。
The drawing is a graph showing the pressure increase during liquid passage when a column is filled with cyanogen bromide-activated granular porous chitin according to the present invention and conventional cyanogen bromide-activated granular agarose.
Claims (1)
状多孔質キチンとし、アルカリ条件下でハロゲン
化シアンと反応させることを特徴とするハロゲン
化シアン活性化粒状多孔質キチンの製造法。1. A method for producing cyanogen halide-activated granular porous chitin, which comprises N-acetylating granular porous chitosan to obtain granular porous chitin, and reacting it with cyanogen halide under alkaline conditions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62073439A JPS63241002A (en) | 1987-03-27 | 1987-03-27 | Preparation of cyanogen halide-activated granular, porous chitin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62073439A JPS63241002A (en) | 1987-03-27 | 1987-03-27 | Preparation of cyanogen halide-activated granular, porous chitin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63241002A JPS63241002A (en) | 1988-10-06 |
| JPH0211601B2 true JPH0211601B2 (en) | 1990-03-15 |
Family
ID=13518275
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62073439A Granted JPS63241002A (en) | 1987-03-27 | 1987-03-27 | Preparation of cyanogen halide-activated granular, porous chitin |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63241002A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114790253B (en) * | 2021-01-25 | 2024-03-26 | 中科南京绿色制造产业创新研究院 | Acylated chitin and preparation method and application thereof |
-
1987
- 1987-03-27 JP JP62073439A patent/JPS63241002A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS63241002A (en) | 1988-10-06 |
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