JPH0145358B2 - - Google Patents
Info
- Publication number
- JPH0145358B2 JPH0145358B2 JP12492081A JP12492081A JPH0145358B2 JP H0145358 B2 JPH0145358 B2 JP H0145358B2 JP 12492081 A JP12492081 A JP 12492081A JP 12492081 A JP12492081 A JP 12492081A JP H0145358 B2 JPH0145358 B2 JP H0145358B2
- Authority
- JP
- Japan
- Prior art keywords
- nad
- transglutaminase
- reaction
- protein
- immobilized
- 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
Links
- 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 claims description 33
- 229950006238 nadide Drugs 0.000 claims description 30
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 30
- 108060008539 Transglutaminase Proteins 0.000 claims description 14
- 102000004169 proteins and genes Human genes 0.000 claims description 14
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- 102000003601 transglutaminase Human genes 0.000 claims description 14
- 239000005515 coenzyme Substances 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 239000007857 degradation product Substances 0.000 claims description 3
- 230000017854 proteolysis Effects 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 description 14
- 235000018102 proteins Nutrition 0.000 description 13
- 102000011632 Caseins Human genes 0.000 description 7
- 108010076119 Caseins Proteins 0.000 description 7
- 239000005018 casein Substances 0.000 description 7
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 7
- 235000021240 caseins Nutrition 0.000 description 7
- 239000000243 solution Substances 0.000 description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 5
- 239000001110 calcium chloride Substances 0.000 description 5
- 229910001628 calcium chloride Inorganic materials 0.000 description 5
- 125000002252 acyl group Chemical group 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- -1 that is Chemical compound 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 238000000354 decomposition reaction Methods 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 2
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 2
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- 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 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000003638 chemical reducing agent Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000011084 recovery Methods 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 1
- 229920000936 Agarose Polymers 0.000 description 1
- 102000009366 Alpha-s1 casein Human genes 0.000 description 1
- 108050000244 Alpha-s1 casein Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 108020000290 Mannitol dehydrogenase Proteins 0.000 description 1
- 108010064851 Plant Proteins Proteins 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 238000003916 acid precipitation Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 235000021120 animal protein Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 229940040511 liver extract Drugs 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 229940101270 nicotinamide adenine dinucleotide (nad) Drugs 0.000 description 1
- 235000021118 plant-derived protein Nutrition 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 229950008679 protamine sulfate Drugs 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 235000019710 soybean protein Nutrition 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 238000006276 transfer reaction Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
本発明はトランスグルタミナーゼを用いて補酵
素ニコチンアミド−アデニン−ジヌクレオチド
（NADと略す）をタンパク質物質に結合させて固
定化したNADを製造する方法に関する。
NADは、酸化還元反応に関係する酵素系にお
いて補酵素として働き、極めて不安定な化合物で
使用時、保存時に変化しやすく、また高価なもの
である。従つて、使用したNADを反応系から分
離、回収し再利用する工夫が必要である。
そこで、本発明者らは、NADを固定化するこ
とを検討した結果、トランスグルタミナーゼを使
用すると首尾よく目的を達成することを見出し
た。本発明は、この知見に基づき完成されたもの
で、固定化された補酵素NADの製法にかかり、
トランスグルタミナーゼを作用させてタンパク質
又はタンパク質分解物にNAD、すなわちニコチ
ンアミド−アデニン−ジヌクレオチド類を結合す
ることを特徴とするものである。
一般に補酵素NAD類を化学的に固定化する方
法は知られているが、NAD類をトランスグルタ
ミナーゼで酵素的に固定化することは、これまで
報告されていない。
本発明によれば、補酵素NADの酵素的固定化
により、NADの回収、再使用が容易になり、ま
た、不安定なNADを安定化した形で得ることが
できる。本発明で得られた固定化NADは、医薬
品などの有用物質を生産する酵素反応や有害物質
を分解、除去する酵素反応において、繰返し使用
することができるから、工業上極めて有利であ
る。
本発明ではNAD又は（及び）NAD誘導体が用
いられ、NADの誘導体としては、例えばアミノ
ヘキシルNAD誘導体すなわち８−（６−アミノヘ
キシル）−NADやN6−〔（６−アミノヘキシル）
カルバモイルメチル〕−NAD等がある。
本発明で使用されるタンパク質又はタンパク質
分解物はNADを固定化する性能、すなわちグル
タミン残基を有するものならば制限はない。タン
パク質として例えば、カゼイン、大豆タンパク質
など全ての動植物タンパク質、菌体タンパク質が
挙げられる。タンパク質分解物としては上記の全
てのタンパク質の分解物を例示することができ
る。またグルタミン残基を含むアミノ酸の合成ポ
リマーも使用しうる。
トランスグルタミナーゼは、カルシウム依存性
のアシル転移反応を触媒する酵素であり、タンパ
ク質又はタンパク質分解物中のグルタミン残基
（アシル供与体）と種々の化合物中のアミノ基
（アシル供与体）と種々の化合物中のアミノ基
（アシル受容体）との間にアミド結合を形成させ
る能力をもつ。トランスグルタミナーゼはコネラ
ンらの方法〔Connellan et al J.Biol.Chem.246、
1093（1971）〕に従つてモルモツトの肝臓から取得
され、他に牛などの血液からも得られる。
本発明の方法ではタンパク質等とNAD類とを
混合したものにトランスグルタミナーゼを作用さ
せて酵素反応を起させタンパク質等とNAD類を
結合させる。一般に、タンパク質１〜５部を水
1000部に混合した液にNAD誘導体0.1〜１部、塩
化カルシウム0.6部、トランスグルタミナーゼ0.1
〜0.2部を添加する。反応はPH６〜8.5、好ましく
は7.5で、温度20〜40℃、好ましくは37℃前後で
行う。反応時間は１〜10時間である。反応時間経
過後、エチレンジアミン四酢酸12部を加え反応を
停止させる。反応終了後、反応液を通常の方法例
えば透析処理して目的物を取得する。
還元剤、例えばシステイン、グルタチオン、メ
ルカプトエタノール、ジチオスレイトールなどは
トランスグルタミナーゼを活性化し、また安定化
する。従つて反応液に還元剤を添加するのが望ま
しいが、必ずしも必須ではない。
好ましい実施態様の１例は下記のとおりであ
る。
トリス緩衝液（PH7.5） 100ｍＭ
塩化カルシウム ５ｍＭ
NAD １ｍＭ
カゼイン １〜３mg／ml
トランスグルタミナーゼ 183μｇ／ml
PH：7.5、反応温度37℃
次に、タンパク質としてカゼインを例にとつ
て、カゼイン中の種々のタンパク画分とNADと
の結合量を検討した結果を述べる。この結合量は
馬肝のアルコール脱水素酵素の補酵素活性により
測定され、測定結果はアセチル化αS1カゼイン画
分の場合が最も効率よくNADと結合することを
示している。
The present invention relates to a method for producing immobilized NAD by binding a coenzyme nicotinamide-adenine dinucleotide (NAD) to a protein substance using transglutaminase. NAD acts as a coenzyme in enzyme systems involved in redox reactions, is an extremely unstable compound, easily changes during use and storage, and is expensive. Therefore, it is necessary to devise ways to separate, recover, and reuse the used NAD from the reaction system. Therefore, the present inventors investigated the immobilization of NAD and found that the purpose could be successfully achieved by using transglutaminase. The present invention was completed based on this knowledge, and involves a method for producing immobilized coenzyme NAD.
It is characterized by binding NAD, that is, nicotinamide-adenine dinucleotides, to proteins or protein degradation products by the action of transglutaminase. Although methods for chemically immobilizing coenzymes NAD are generally known, enzymatic immobilization of NAD using transglutaminase has not been reported so far. According to the present invention, enzymatic immobilization of the coenzyme NAD facilitates recovery and reuse of NAD, and also allows unstable NAD to be obtained in a stabilized form. The immobilized NAD obtained in the present invention is industrially extremely advantageous because it can be used repeatedly in enzymatic reactions to produce useful substances such as pharmaceuticals and in enzyme reactions to decompose and remove harmful substances. In the present invention, NAD or (and) a NAD derivative is used, and examples of the derivative of NAD include aminohexyl NAD derivatives, i.e., 8-(6-aminohexyl)-NAD and N 6 -[(6-aminohexyl)
carbamoylmethyl]-NAD, etc. The protein or protein decomposition product used in the present invention is not limited as long as it has the ability to immobilize NAD, that is, it has a glutamine residue. Examples of proteins include all animal and plant proteins such as casein and soybean protein, and bacterial cell proteins. Examples of protein decomposition products include all of the above-mentioned protein decomposition products. Synthetic polymers of amino acids containing glutamine residues may also be used. Transglutaminase is an enzyme that catalyzes a calcium-dependent acyl transfer reaction between glutamine residues (acyl donors) in proteins or protein degradation products and amino groups (acyl donors) in various compounds. It has the ability to form an amide bond with the amino group (acyl acceptor) inside. Transglutaminase was determined by the method of Connellan et al [Connellan et al J.Biol.Chem.246,
1093 (1971)] from the liver of guinea pigs, and can also be obtained from the blood of cows and other animals. In the method of the present invention, transglutaminase is applied to a mixture of proteins, etc. and NADs to cause an enzymatic reaction, thereby binding the proteins, etc. and NADs. Generally, mix 1 to 5 parts of protein with water.
0.1 to 1 part of NAD derivative, 0.6 part of calcium chloride, and 0.1 part of transglutaminase to 1000 parts of mixed solution.
Add ~0.2 part. The reaction is carried out at a pH of 6 to 8.5, preferably 7.5, and a temperature of 20 to 40°C, preferably around 37°C. Reaction time is 1 to 10 hours. After the reaction time has elapsed, 12 parts of ethylenediaminetetraacetic acid is added to stop the reaction. After the reaction is completed, the reaction solution is subjected to a conventional method such as dialysis treatment to obtain the target product. Reducing agents such as cysteine, glutathione, mercaptoethanol, dithiothreitol, etc. activate and also stabilize transglutaminase. Therefore, it is desirable, but not essential, to add a reducing agent to the reaction solution. An example of a preferred embodiment is as follows. Tris buffer (PH7.5) 100mM Calcium chloride 5mM NAD 1mM Casein 1-3mg/ml Transglutaminase 183μg/ml PH: 7.5, reaction temperature 37℃ We will describe the results of examining the amount of binding between protein fractions and NAD. The amount of this binding is measured by the coenzyme activity of horse liver alcohol dehydrogenase, and the measurement results show that the acetylated αS1 casein fraction binds to NAD most efficiently.
【表】
本発明で得られた固定化NADを例えば医薬品
等製造の酸化反応に使用すると、NADは還元型
NAD（NADH）に変るが、ここに生じたNADH
−タンパク質固定化結合体は、塩化カルシウム等
による塩析又は酸沈殿によつて反応液から容易に
分離、回収することが可能なので、NADを有効
に再使用することができる。この際の回収率はほ
ぼ100％である。また、固定化NADは遊離のもの
に比し保存安定性に優れている。
次に、本発明を実施例によつて説明する。
実施例
〔トランスグルタミナーゼの製法〕
モルモツト肝臓の抽出液を出発原料として
DEAE−セルロースによるクロマトグラフイー、
プロタミン硫酸分画、アガロースによるゲルクロ
マトグラフイーを行い、トランスグルタミナーゼ
を精製した。２Kgの肝臓より200mgの精製酵素を
得た。
〔固定化NADの製法〕
塩化カルシウム0.6ｇを溶解したトリス塩酸緩
衝液（PH7.5）１中に、カゼインを３ｇおよび
アミノヘキシルNAD誘導体0.8ｇを添加、溶解し
て反応液とした。
これにトランスグルタミナーゼを200mg加え、
37℃で８時間反応させた。
反応は0.4MのEDTA（PH8.0）270mlを加えて停
止させた。
反応液を70ｍＭ KClを含む10ｍＭイミダゾー
ル緩衝液（PH6.0）に透析して、遊離のNAD誘導
体を除き、NAD誘導体・カゼイン複合体2.8ｇを
得た。
使用例
Ｄ−フラクトースの前駆体Ｄ−マニトールを原
料としてマニトールデヒドロゲナーゼと、実施例
１で得たNAD誘導体・カゼイン複合体を共用し
て酸化反応させ、Ｄ−フラクトースを得た。
反応終了後、反応液に塩化カルシウムを最終濃
度が40〜60ｍＡになるようにカゼイン複合体を沈
殿させて、分離、回収した。
この回収したNADH誘導体・カゼイン複合体
はアルコールデヒドロゲナーゼと共用して例えば
アルデヒド類からアルコール類を得ることができ
る。[Table] When the immobilized NAD obtained in the present invention is used, for example, in an oxidation reaction in the production of pharmaceuticals, NAD is in the reduced form.
It changes to NAD (NADH), but the NADH generated here
- Since the protein-immobilized conjugate can be easily separated and recovered from the reaction solution by salting out with calcium chloride or the like or acid precipitation, NAD can be effectively reused. The recovery rate in this case is almost 100%. Furthermore, immobilized NAD has superior storage stability compared to free NAD. Next, the present invention will be explained with reference to examples. Example [Production method of transglutaminase] Using guinea pig liver extract as a starting material
DEAE-cellulose chromatography,
Transglutaminase was purified by protamine sulfate fractionation and gel chromatography using agarose. 200 mg of purified enzyme was obtained from 2 kg of liver. [Production method of immobilized NAD] 3 g of casein and 0.8 g of aminohexyl NAD derivative were added and dissolved in 1 part of Tris-HCl buffer (PH7.5) in which 0.6 g of calcium chloride was dissolved to prepare a reaction solution. Add 200mg of transglutaminase to this,
The reaction was carried out at 37°C for 8 hours. The reaction was stopped by adding 270 ml of 0.4M EDTA (PH8.0). The reaction solution was dialyzed against 10mM imidazole buffer (PH6.0) containing 70mM KCl to remove free NAD derivatives, yielding 2.8g of NAD derivative/casein complex. Example of Use Using D-mannitol, a precursor of D-fructose, as a raw material, an oxidation reaction was carried out using mannitol dehydrogenase and the NAD derivative/casein complex obtained in Example 1 to obtain D-fructose. After the reaction was completed, calcium chloride was added to the reaction solution to a final concentration of 40 to 60 mA to precipitate the casein complex, which was then separated and recovered. The recovered NADH derivative/casein complex can be used together with alcohol dehydrogenase to obtain alcohols from aldehydes, for example.
Claims (1)
ク質又はタンパク質分解物に補酵素ニコチンアミ
ド−アデニン−ジヌクレオチド類を結合すること
を特徴とする固定化補酵素の製法。1. A method for producing an immobilized coenzyme, which comprises binding a coenzyme nicotinamide-adenine dinucleotide to a protein or protein degradation product by the action of transglutaminase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP56124920A JPS5828295A (en) | 1981-08-10 | 1981-08-10 | Preparation of immobilized coenzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP56124920A JPS5828295A (en) | 1981-08-10 | 1981-08-10 | Preparation of immobilized coenzyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5828295A JPS5828295A (en) | 1983-02-19 |
| JPH0145358B2 true JPH0145358B2 (en) | 1989-10-03 |
Family
ID=14897402
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP56124920A Granted JPS5828295A (en) | 1981-08-10 | 1981-08-10 | Preparation of immobilized coenzyme |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5828295A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5834232A (en) * | 1996-05-01 | 1998-11-10 | Zymogenetics, Inc. | Cross-linked gelatin gels and methods of making them |
-
1981
- 1981-08-10 JP JP56124920A patent/JPS5828295A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5828295A (en) | 1983-02-19 |
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