JPH0249711B2 - KOBUNSHINISEITAIKOBUNSHIOKETSUGOSASERUHOHO - Google Patents
KOBUNSHINISEITAIKOBUNSHIOKETSUGOSASERUHOHOInfo
- Publication number
- JPH0249711B2 JPH0249711B2 JP11465883A JP11465883A JPH0249711B2 JP H0249711 B2 JPH0249711 B2 JP H0249711B2 JP 11465883 A JP11465883 A JP 11465883A JP 11465883 A JP11465883 A JP 11465883A JP H0249711 B2 JPH0249711 B2 JP H0249711B2
- Authority
- JP
- Japan
- Prior art keywords
- polymer
- biopolymer
- activated
- amino group
- reaction
- 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
- 229920000642 polymer Polymers 0.000 claims description 29
- 229920001222 biopolymer Polymers 0.000 claims description 27
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 18
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- YBBRCQOCSYXUOC-UHFFFAOYSA-N sulfuryl dichloride Chemical compound ClS(Cl)(=O)=O YBBRCQOCSYXUOC-UHFFFAOYSA-N 0.000 claims description 7
- 125000000467 secondary amino group Chemical group [H]N([*:1])[*:2] 0.000 claims description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 12
- 230000000694 effects Effects 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 9
- 229920002678 cellulose Polymers 0.000 description 9
- 239000001913 cellulose Substances 0.000 description 9
- 239000011324 bead Substances 0.000 description 8
- 150000004676 glycans Chemical class 0.000 description 7
- 229920001282 polysaccharide Polymers 0.000 description 7
- 239000005017 polysaccharide Substances 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 6
- 108010093096 Immobilized Enzymes Proteins 0.000 description 5
- -1 cyanogen halide Chemical class 0.000 description 5
- JMANVNJQNLATNU-UHFFFAOYSA-N glycolonitrile Natural products N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 description 5
- 102100036504 Dehydrogenase/reductase SDR family member 9 Human genes 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- DJQSZCMOJNOVPS-UHFFFAOYSA-N 1h-imidazol-3-ium-3-sulfonate Chemical class OS(=O)(=O)N1C=CN=C1 DJQSZCMOJNOVPS-UHFFFAOYSA-N 0.000 description 3
- 101000928746 Homo sapiens Dehydrogenase/reductase SDR family member 9 Proteins 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 3
- 238000009739 binding Methods 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 239000004380 Cholic acid Substances 0.000 description 2
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 235000019416 cholic acid Nutrition 0.000 description 2
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 2
- 229960002471 cholic acid Drugs 0.000 description 2
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 description 2
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 2
- 235000011180 diphosphates Nutrition 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 101710172561 3alpha-hydroxysteroid dehydrogenase Proteins 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 239000004354 Hydroxyethyl cellulose Substances 0.000 description 1
- 229920000663 Hydroxyethyl cellulose Polymers 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003613 bile acid Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- OSQPUMRCKZAIOZ-UHFFFAOYSA-N carbon dioxide;ethanol Chemical compound CCO.O=C=O OSQPUMRCKZAIOZ-UHFFFAOYSA-N 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 235000019447 hydroxyethyl cellulose Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229950006238 nadide Drugs 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- 238000010534 nucleophilic substitution reaction Methods 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 125000000472 sulfonyl group Chemical group *S(*)(=O)=O 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Description
【発明の詳細な説明】
本発明はセルロース等の水酸基を有する高分子
に酵素、蛋白質等のアミノ基を有する生体高分子
を結合させる方法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for bonding a biopolymer having an amino group such as an enzyme or protein to a polymer having a hydroxyl group such as cellulose.
従来より、酵素や蛋白質を担体である高分子物
質に化学的に結合させて固定化した固定化酵素等
をバイオリアクターや液体クロマトグラフイー用
カラム充填剤として利用することが行われてい
る。例えば１個以上の水酸基を有する多糖類など
の高分子に好ましくはPH8〜13のアルカリ性条
件下でハロゲン化シアンを作用させることにより
活性化し、次にこれにアミノ基を有する生体高分
子を弱アルカリ性条件で反応させて結合させる方
法や多糖高分子の一種であるアガロースに塩化Ｐ
−トリエンスルホニルを作用させて活性化し、こ
れに求核置換反応によつてペルオキシダーゼやア
ルコールデヒドロゲナーゼをそれぞれ18％及び25
％程度の活性固定化率で固定化することが知られ
ている。 BACKGROUND ART Conventionally, immobilized enzymes, etc., in which enzymes and proteins are chemically bonded and immobilized to polymeric substances as carriers, have been used as column packing materials for bioreactors and liquid chromatography. For example, a polymer such as a polysaccharide having one or more hydroxyl groups is preferably activated by acting on a cyanogen halide under alkaline conditions of pH 8 to 13, and then a biopolymer having an amino group is activated under weakly alkaline conditions. A method of bonding by reacting under certain conditions, and a method of combining P chloride with agarose, a type of polysaccharide polymer.
-activated by the action of trienesulfonyl, and activated by a nucleophilic substitution reaction to convert peroxidase and alcohol dehydrogenase to 18% and 25%, respectively.
It is known that the activity is immobilized at an activity immobilization rate of about 1.5%.
しかしながら、上記のうちハロゲン化シアンを
用いる方法においては、該ハロゲン化シアンは猛
毒であるので取扱いに注意を要し、使用後の排水
の処理にも手間がかかり、又、活性化された高分
子と生体高分子との反応はアルカリ性で行う必要
があるので、アルカリ条件下で失活するような生
体高分子は使用出来ないという欠点があり、後者
の場合については活性固定化率が低いという欠点
がある。 However, among the above methods using cyanogen halides, the cyanogen halides are highly poisonous, so they must be handled with care, and it takes time and effort to treat wastewater after use. Since the reaction between biopolymer and biopolymer must be carried out under alkaline conditions, it has the disadvantage that biopolymers that are deactivated under alkaline conditions cannot be used, and in the latter case, the activity immobilization rate is low. There is.
本発明は上記の如き現状にかんがみ、ハロゲン
化シアンの如き有毒な試薬を用いずともよく、さ
らにアルカリ性の条件下で失活する様な生体高分
子を失活させることなく高分子担体に結合させる
ことが出来、かつ高い活性固定化率で生体高分子
を水酸基を有する高分子に化学的に結合すること
の出来る方法を提供することを目的としてなされ
たものであり、その要旨は第１級又は第２級のア
ミノ基を含有する生体高分子を共有結合により水
酸基を含有する高分子に結合させる方法におい
て、上記高分子を塩化スルフリルと、次いでイミ
ダゾールと反応させたのち、これを上記生体高分
子と反応させることを特徴とする高分子に生体高
分子を結合させる方法に存する。 In view of the above-mentioned current situation, the present invention eliminates the need to use toxic reagents such as cyanogen halides, and furthermore allows biopolymers that would be deactivated under alkaline conditions to be bonded to a polymer carrier without being deactivated. The purpose of this research is to provide a method for chemically bonding biopolymers to polymers having hydroxyl groups at a high activity immobilization rate. In a method for covalently bonding a biopolymer containing a secondary amino group to a polymer containing a hydroxyl group, the polymer is reacted with sulfuryl chloride and then with imidazole, and then the biopolymer is The invention consists in a method of bonding a biopolymer to a polymer characterized by reacting with a biopolymer.
本発明において用いられる水酸基を含有する高
分子としては、多糖類やその誘導体が挙げられ、
例えばデキストラン、セルロース、デンプン、デ
キストリン、アガロースなどの多糖類や、ヒドロ
キシエチルセルロースの様な多糖類の水酸基含有
誘導体が好適に用いられる。 Polymers containing hydroxyl groups used in the present invention include polysaccharides and derivatives thereof,
For example, polysaccharides such as dextran, cellulose, starch, dextrin, and agarose, and hydroxyl group-containing derivatives of polysaccharides such as hydroxyethylcellulose are preferably used.
本発明にもとづいて、生体物質を多糖類等の水
酸基を含有する高分子担体に結合させるには、ま
ず、該高分子を塩化スルフリルと反応させて、高
分子の水酸基を式（）の如く変化させ、
Ｒ−OH塩化スルフリル
――――――――→
Ｒ−OSO2・Cl （）
次いで、これにイミダゾールを加えて反応し
て、式（）の如く１−イミダゾリル−スルホナ
ート誘導体を形成させ、
高分子の水酸基を活性化させる。（Ｒは高分子
残基）
なお式（）の反応は通常、水分を十分除いた
高分子を、Ｎ，Ｎ−ジメチルホルムアミド等の適
宜な有機媒質に懸濁若しくは溶解させ、−40〜−
50℃に冷却させた状態で塩化スルフリルを滴下し
て行うのが好ましい。 Based on the present invention, in order to bond a biological material to a polymer carrier containing a hydroxyl group such as a polysaccharide, the polymer is first reacted with sulfuryl chloride to change the hydroxyl group of the polymer as shown in the formula (). and R-OH sulfuryl chloride――――――――→ R-OSO 2 Cl () Next, imidazole is added to this and reacted to form a 1-imidazolyl-sulfonate derivative as shown in formula (). , Activates the hydroxyl groups of polymers. (R is a polymer residue) The reaction of formula () is usually carried out by suspending or dissolving a polymer from which moisture has been sufficiently removed in an appropriate organic medium such as N,N-dimethylformamide,
It is preferable to drop sulfuryl chloride in a state cooled to 50°C.
又、式（）の反応は、上記において塩化スル
フリルを滴下したのち、大過剰のイミダゾールを
加えて、徐々に温度を室温まで上昇させながら行
うのが好ましい。 Further, the reaction of formula () is preferably carried out by adding a large excess of imidazole after dropping sulfuryl chloride as described above, and gradually raising the temperature to room temperature.
かくして生成した１−イミダゾリル−スルホナ
ート誘導体は生体高分子中のアミノ基と中性附近
で式（）の如くに反応して、
（は生体高分子残基を示す）
共有結合によつて安定に結合した高分子−生体
高分子結合物を与える。 The 1-imidazolyl-sulfonate derivative thus produced reacts with the amino group in the biopolymer near neutrality as shown in formula (), (denotes a biopolymer residue) Provides a polymer-biopolymer conjugate stably bound by covalent bonds.
なお、上式（）では第１級アミノ基（−
NH2）を有する生体高分子が活性化された高分
子担体と結合する機構を示したが、第２級アミノ
基（NH）を有する生体高分子についても、上
式（）と同様の機構で活性化高分子と結合す
る。 In addition, in the above formula (), the primary amino group (-
Although we have shown the mechanism by which a biopolymer having a secondary amino group (NH 2 ) binds to an activated polymer carrier, the same mechanism as in the above formula () can be used for a biopolymer having a secondary amino group (NH). Combines with activated polymer.
従つて、本発明においては、結合せんとする第
１級又は第２級のアミノ基を有する例えば酵素、
蛋白質等の生体高分子を中性附近のPH値を有す
る媒質、好ましくは緩衝液に溶解したものに、上
記で用意した水酸基を含有する高分子の活性化物
すなわち１−イミダゾリルスルホナート誘導体を
加えて撹拌すれば上記結合物を得ることが出来
る。 Therefore, in the present invention, for example, an enzyme having a primary or secondary amino group to be bound,
A biopolymer such as a protein is dissolved in a medium having a pH value around neutrality, preferably a buffer solution, and the activated product of the polymer containing a hydroxyl group, that is, the 1-imidazolylsulfonate derivative prepared above, is added. The above-mentioned combined product can be obtained by stirring.
又、本発明においては、上記の如くして生体高
分子を結合させたのち、セルロース等の高分子に
なお残存する活性点をブロツクしておくのが好ま
しく、このためには、0.05％程度の２−メルカプ
トエタノールを含む約PH8のトリス塩酸緩衝液
に浸漬すればよい。 In addition, in the present invention, after binding biopolymers as described above, it is preferable to block active sites still remaining in the polymer such as cellulose. It may be immersed in a Tris-HCl buffer solution containing 2-mercaptoethanol and having a pH of about 8.
上記の如くして本発明にもとづいて用意された
多糖類等の高分子と酵素や蛋白質等の生体高分子
との結合物は、固定化酵素等としてバイオリアク
ターや、胆汁酸等の生体物質分析用の液体クロマ
トグラフイーに用いられる固定化酵素による反応
カラムの充填剤として使用出来るのであり、この
使用に際して酵素等生体高分子は高分子担体に共
有結合によつて強固に結合しているので簡単に分
解されず、広いPH範囲においても安定に使用さ
れ得るのである。 The combination of a polymer such as a polysaccharide and a biopolymer such as an enzyme or a protein prepared according to the present invention as described above can be used as an immobilized enzyme in a bioreactor or for analysis of biological substances such as bile acids. It can be used as a packing material for reaction columns with immobilized enzymes used in liquid chromatography, and it is easy to use because enzymes and other biopolymers are firmly bound to the polymer carrier by covalent bonds. It does not decompose and can be used stably even in a wide pH range.
本発明方法は上述の通りの方法であり、とく
に、１個以上の水酸基を含有する高分子を塩化ス
ルフリルと、次いでイミダゾールと反応させて活
性化したのち、これを第１級又は第２級アミノ基
を含有する生体高分子と反応させ、上記高分子と
生体高分子とを共有結合により結合させる方法で
あるので、本発明方法によれば、従来法の如く毒
性の高いハロゲン化シアンなどの試薬を用いずと
もよく、安全に反応を行うことが出来、又、結合
反応を中性の条件で行い得るのでアルカリ性で失
活する様な生体高分子についても高分子担体に結
合させて利用することが出来、しかも高い活性固
定化率で高分子担体に酵素等の生体高分子を結合
させることが出来るという効果を奏するのであ
る。 The method of the present invention is as described above, and in particular, after activating a polymer containing one or more hydroxyl groups with sulfuryl chloride and then with imidazole, Since the method of the present invention involves reacting with a biopolymer containing a group and covalently bonding the polymer and the biopolymer, unlike conventional methods, highly toxic reagents such as cyanogen halides are not used. The reaction can be carried out safely without the need to use a polymer, and since the binding reaction can be carried out under neutral conditions, biopolymers that are inactivated by alkalinity can also be used by binding them to a polymer carrier. Moreover, it is possible to bind biopolymers such as enzymes to the polymer carrier with a high activity immobilization rate.
以下本発明につき、実施例にもとづいて説明す
る。 The present invention will be explained below based on examples.
実施例
担体の活性化：セルロースビーズ（商品名セル
ロフアイン700M、生化学工業社製）を蒸留水で
洗浄後、脱水したＮ，Ｎ−ジメチルホルムアミド
（DMF）中で平衡化したものから、セルロースビ
ーズ2.6ｇを取り出し、DMF60mlに撹拌、懸濁
し、ドライアイス−エタノール浴で−40℃に冷却
した。ゆるやかに撹拌しながらこれに塩化スルフ
リル1.6mlを滴下すると反応液は淡黄色になつた。
30分後、イミダゾール10.8ｇを添加し、撹拌しな
がら室温に戻し、セルロースビーズを別して取
り出し、DMF、蒸留水、DMFの順に洗浄し、吸
引脱水を十分に行つて活性化セルロースビーズを
用意した。Example Activation of carrier: Cellulose beads (trade name: Cellulofine 700M, manufactured by Seikagaku Corporation) were washed with distilled water and then equilibrated in dehydrated N,N-dimethylformamide (DMF). Cellulose beads 2.6 g was taken out, stirred and suspended in 60 ml of DMF, and cooled to -40°C in a dry ice-ethanol bath. When 1.6 ml of sulfuryl chloride was added dropwise to the mixture with gentle stirring, the reaction liquid turned pale yellow.
After 30 minutes, 10.8 g of imidazole was added, and the mixture was returned to room temperature with stirring, and the cellulose beads were taken out separately, washed with DMF, distilled water, and DMF in this order, and sufficiently dehydrated by suction to prepare activated cellulose beads.
酵素の固定化：次に、3α−ヒドロキシステロ
イドデヒドロゲナーゼ（3α−HSD）12.6mgを
PH7.0の0.1リン酸緩衝液２mlにとかしたものに、
上記で用意した活性化セルロースビーズ0.62ｇを
加え、撹拌しながら室温にて反応させた。２時間
後別し、セルロースビーズ上に残つている活性
点をブロツクするため、0.05％の２−メルカプト
エタノールを含むPH8.0の0.1Mトリス塩酸緩衝
液に浸漬した。４℃にて２時間静置後、別し、
0.05％の２−メルカプトエタノール及び0.1mMの
EDTAを含むPH7.0の100mMリン酸緩衝液中に
保存した。 Enzyme immobilization: Next, 12.6 mg of 3α-hydroxysteroid dehydrogenase (3α-HSD) was added.
Dissolved in 2 ml of 0.1 phosphate buffer with pH 7.0,
0.62 g of the activated cellulose beads prepared above were added and reacted at room temperature with stirring. After 2 hours, the beads were separated and immersed in 0.1M Tris-HCl buffer at pH 8.0 containing 0.05% 2-mercaptoethanol to block active sites remaining on the beads. After standing at 4℃ for 2 hours, separate,
0.05% 2-mercaptoethanol and 0.1mM
Stored in 100mM phosphate buffer, pH 7.0, containing EDTA.
固定化率の算出：かくして得られた3α−HSD
固定化セルロースビーズの固定化率を調べるた
め、上記3α−HSD溶液の固定化に用いる前と固
定化後における酵素活性を、PH9.5の20mMピロ
リン酸緩衝液（25℃）中で、補酵素ニコチンアミ
ドアデニンジヌクレオチド及び基質（コール酸）
の存在下に測定すると、固定化前の酵素活性は
0.275ユニツト／0.1ml、固定化後の酵素活性は
0.0174ユニツト／0.1mlであり、従つて固定化率
は93.7％と算出された。 Calculation of immobilization rate: 3α-HSD thus obtained
In order to investigate the immobilization rate of the immobilized cellulose beads, the enzyme activity before and after immobilization of the above 3α-HSD solution was measured in a 20mM pyrophosphate buffer (25°C) with a pH of 9.5. Nicotinamide adenine dinucleotide and substrate (cholic acid)
When measured in the presence of
0.275 units/0.1ml, enzyme activity after immobilization is
It was 0.0174 units/0.1 ml, so the immobilization rate was calculated to be 93.7%.
固定化酵素の活性測定：PH9.5の20mMピロリ
ン酸緩衝液（25℃）中で、補酵素ニコチンアミド
アデニンヌクレオチド1mM及び基質（コール酸）
1mMの存在下に、精秤した固定化酵素を添加、
撹拌して反応させ、活性を調べた所、8.9ユニツ
ト／ｇの比活性値が測定された。 Activity measurement of immobilized enzyme: Coenzyme nicotinamide adenine nucleotide 1mM and substrate (cholic acid) in 20mM pyrophosphate buffer (25℃) at pH 9.5.
Add precisely weighed immobilized enzyme in the presence of 1mM,
When the reaction was stirred and the activity was examined, a specific activity value of 8.9 units/g was measured.
Claims (1)
高分子を共有結合により、水酸基を含有する高分
子に結合させる方法において、上記高分子を塩化
スルフリルと、次いでイミダゾールと反応させた
のち、これを上記生体高分子と反応させることを
特徴とする高分子に生体高分子を結合させる方
法。1. In a method for covalently bonding a biopolymer containing a primary or secondary amino group to a polymer containing a hydroxyl group, the polymer is reacted with sulfuryl chloride and then with imidazole, and then A method for bonding a biopolymer to a polymer, the method comprising reacting this with the biopolymer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11465883A JPH0249711B2 (en) | 1983-06-24 | 1983-06-24 | KOBUNSHINISEITAIKOBUNSHIOKETSUGOSASERUHOHO |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11465883A JPH0249711B2 (en) | 1983-06-24 | 1983-06-24 | KOBUNSHINISEITAIKOBUNSHIOKETSUGOSASERUHOHO |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS606703A JPS606703A (en) | 1985-01-14 |
| JPH0249711B2 true JPH0249711B2 (en) | 1990-10-31 |
Family
ID=14643315
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11465883A Expired - Lifetime JPH0249711B2 (en) | 1983-06-24 | 1983-06-24 | KOBUNSHINISEITAIKOBUNSHIOKETSUGOSASERUHOHO |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0249711B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016098313A (en) * | 2014-11-21 | 2016-05-30 | セイコーエプソン株式会社 | Cellulose-based material, liquid composition, molded object, and method for manufacturing molded object |
-
1983
- 1983-06-24 JP JP11465883A patent/JPH0249711B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS606703A (en) | 1985-01-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4175073A (en) | Reactive derivatives of HS-group-containing polymers | |
| US4542102A (en) | Coupling of nucleic acids to solid support by photochemical methods | |
| US4330440A (en) | Activated matrix and method of activation | |
| US5092992A (en) | Polyethyleneimine matrixes for affinity chromatography | |
| Suekane | Immobilization of glucose isomerase | |
| EP0130523B1 (en) | Immobilized nucleic acid probe and solid support for nucleic acids | |
| DE2247163A1 (en) | CARRIER MATRIX FOR FIXING BIOLOGICALLY ACTIVE SUBSTANCES AND THE PROCESS FOR THEIR PRODUCTION | |
| US6004786A (en) | Inorganic carrier containing bound silane coupling agent having carboxylic-ester group for immobilizing lipase | |
| JPS6317841B2 (en) | ||
| EP0012751A4 (en) | Preparation of trichloro-s-triazine activated supports. | |
| JPH07147981A (en) | Method for production of immobilized enzyme complex and saidimmobilized enzyme complex produced by said method | |
| US5085779A (en) | Polyethyleneimine matrixes for affinity chromatography | |
| Hsiao et al. | Immobilization of glycoenzymes through carbohydrate side chains | |
| HU179727B (en) | Process for producing water-insoluble enzyme composition | |
| EP0487104A1 (en) | Method of immobilizing single-stranded DNA on carrier at terminal | |
| US3278392A (en) | Water insoluble enzymes | |
| JPS6239997B2 (en) | ||
| JPH0249711B2 (en) | KOBUNSHINISEITAIKOBUNSHIOKETSUGOSASERUHOHO | |
| JPH0249712B2 (en) | SHINSUISEIKOBUNSHINISEITAIKOBUNSHIOKETSUGOSASERUHOHO | |
| US4562251A (en) | Agarose derivatives of amino phenyl boronic acid | |
| JPH0551279B2 (en) | ||
| JPH06340707A (en) | Method of activation of polymer carrier | |
| JP3025947B2 (en) | Method for producing dry immobilized lipase carrier | |
| SE463921B (en) | PROCEDURES FOR IMMOBILIZATION OF SOCIETIES INCLUDING NUCLEOPHIL GROUPS | |
| Ibrahim et al. | Immobilization of epoxide hydrolase purified from rat liver microsomes |