JPH047377B2 - - Google Patents
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- Publication number
- JPH047377B2 JPH047377B2 JP59178044A JP17804484A JPH047377B2 JP H047377 B2 JPH047377 B2 JP H047377B2 JP 59178044 A JP59178044 A JP 59178044A JP 17804484 A JP17804484 A JP 17804484A JP H047377 B2 JPH047377 B2 JP H047377B2
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- immobilized
- membrane
- polysulfone
- physiologically active
- 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
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Manufacture Of Macromolecular Shaped Articles (AREA)
- Compositions Of Macromolecular Compounds (AREA)
- Polymers With Sulfur, Phosphorus Or Metals In The Main Chain (AREA)
- Materials For Medical Uses (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、生理活性物質固定化膜の製造法に関
する。更に詳しくは、酵素、微生物などの生理活
性物質を固定化せしめるのに用いられるポリスル
ホン膜の製造法に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing a physiologically active substance-immobilized membrane. More specifically, the present invention relates to a method for producing a polysulfone membrane used for immobilizing physiologically active substances such as enzymes and microorganisms.
従来から、酵素、微生物などの生理活性物質を
種々の膜材料上に固定化し、例えば医用材料、バ
イオリアクター、バイオセンサーなどの用途に用
いることが行われている。膜材料として代表的な
ポリスルホンを用いる場合には、ポリスルホンの
ドープ液に所定の酵素を添加し、その混合液を製
膜した後、酵素架橋試薬であるグルタルアルデヒ
ドの水溶液中に浸漬することによつて、酵素を膜
内部に固定化せしめている。
BACKGROUND ART Bioactive substances such as enzymes and microorganisms have been immobilized on various membrane materials and used for applications such as medical materials, bioreactors, and biosensors. When polysulfone, a typical membrane material, is used, a specified enzyme is added to a polysulfone dope solution, the mixture is formed into a film, and then immersed in an aqueous solution of glutaraldehyde, an enzyme crosslinking reagent. As a result, the enzyme is immobilized inside the membrane.
このような酵素の固定化方法によると、酵素と
比較してグルタルアルデヒド分子の方がきわめて
小さいため、酵素同士の架橋以外に、酵素分子中
に存在するアミノ基同士を架橋させてしまうた
め、酵素活性の失活を招き、活性の低下や不安定
化などがみられるようになる。また、膜材料とし
て用いられるポリスルホンは、酵素を固定化する
のに必要な官能基を有していないため、その固定
化力は更に弱いものとなつている。 According to this enzyme immobilization method, since the glutaraldehyde molecule is extremely small compared to the enzyme, it cross-links the amino groups present in the enzyme molecules in addition to cross-linking the enzymes. This leads to deactivation of activity, resulting in decreased activity and instability. Furthermore, polysulfone used as a membrane material does not have functional groups necessary for immobilizing enzymes, and therefore its immobilizing power is even weaker.
前述の如く、ポリスルホンは代表的な膜材料で
あり、しかもそこに酵素を固定化せしめたものは
各種の有用な用途を有しているので、酵素固定膜
を活性およびその持続性の向上は、それの有用性
を更に高めることが期待される。本発明者は、か
かる課題の解決方法を求めて種々検討の結果、ア
ルデヒド化合物のみによつて酵素を固定化させる
のではなく、その間にスペーサーとしてトリアミ
ン化合物を導入することにより、上記課題が有効
に解決されることを見出した。このような解決方
法はまた、酵素だけではなく、微生物およびその
構造中にアミノ基を有するたん白質などの生物学
的活性を有する他の物質にも適用することができ
る。
As mentioned above, polysulfone is a typical membrane material, and enzymes immobilized thereon have various useful uses.Improving the activity and sustainability of enzyme-immobilized membranes is It is expected that this will further enhance its usefulness. As a result of various studies in search of a solution to this problem, the present inventor found that the above problem can be effectively solved by not immobilizing the enzyme only with an aldehyde compound, but by introducing a triamine compound as a spacer between them. I found a solution. Such a solution can also be applied not only to enzymes, but also to other substances with biological activity, such as microorganisms and proteins that have amino groups in their structure.
〔問題点を解決するための手段〕および〔作用〕
従つて、本発明は生理活性物質固定化膜の製造
法に係り、生理活性物質固定化膜の製造は、ポリ
スルホンのドープ液中にアルデヒド化合物および
トリアミン化合物を添加した混合液を製膜するこ
とによつて行われる。[Means for Solving the Problem] and [Operation] Therefore, the present invention relates to a method for producing a physiologically active substance-immobilized membrane. This is carried out by forming a film from a mixed solution to which a triamine compound is added.
ポリスルホンのドープ液は、ポリスルホンをジ
メチルホルムアミド、ジメチルアセトアミド、ジ
エチルホルムアミド、ジエチルアセトアミド、モ
ルホリン、Ｎ−メチル−２−ピロリドン、テトラ
ヒドロフラン、ジメチルスルホキシド、トリメチ
ルホスフエートなどの溶媒中に、約10〜30重量％
程度の濃度で溶解されることにより調整される。 The polysulfone dope solution is prepared by adding about 10 to 30% by weight of polysulfone in a solvent such as dimethylformamide, dimethylacetamide, diethylformamide, diethylacetamide, morpholine, N-methyl-2-pyrrolidone, tetrahydrofuran, dimethylsulfoxide, or trimethylphosphate.
It is adjusted by dissolving it at a certain concentration.
このポリスルホンドープ液には、グルタルアル
デヒド、ホルムアルデヒド、アセトアルデヒドな
どのアルデヒド化合物および１，８−ジアミノ−
４−アミノメチルオクタンによつて代表されるト
リアミン化合物が、そのままの状態あるいは水溶
液などの溶液状態で、ドープ液10ml当りアルデヒ
ド化合物として約0.2〜5.0ml、またトリアミン化
合物として約0.1〜2.0mlの量で添加される。 This polysulfone dope solution contains aldehyde compounds such as glutaraldehyde, formaldehyde, acetaldehyde, and 1,8-diamino-
A triamine compound represented by 4-aminomethyloctane is contained in an amount of about 0.2 to 5.0 ml as an aldehyde compound and about 0.1 to 2.0 ml as a triamine compound per 10 ml of dope solution, either as it is or in a solution state such as an aqueous solution. It is added in
ポリスルホンドープ液からの製膜は、それをガ
ラス板、フツ素樹脂などの基質上にそれを流延
し、乾燥させる方法、水中に浸漬してゲル化させ
る方法、環状ノズルから水中に紡糸してゲル化さ
せる方法などによつて、平膜状、中空糸状などに
成形することにより行われる。 Films can be formed from polysulfone dope by casting it onto a substrate such as a glass plate or fluororesin and drying it, by immersing it in water to gel it, or by spinning it into water from an annular nozzle. This is done by forming it into a flat membrane shape, hollow fiber shape, etc. by a gelling method or the like.
このようにして製膜されたポリスルホン膜への
生理活性物質の固定は、例えば酵素の場合には一
般に約0.1〜10mg／mlの濃度の水溶液中に浸漬す
ることにより行われる。 For example, in the case of enzymes, physiologically active substances are immobilized on the polysulfone membrane thus formed by immersion in an aqueous solution having a concentration of about 0.1 to 10 mg/ml.
酵素としては、例えばグルコースオキシダー
ゼ、アミノ酸オキシダーゼ、コレステロールオキ
シダーゼ、ウリカーゼなどのオキシダーゼ類、ウ
リアーゼ、クレアキニナーゼ、グルタミナーゼ、
ペニシリナーゼ、カタラーゼ、パーオキシダー
ゼ、インベルターゼ、ムタロターゼ、アミラー
ゼ、パパイン、トリプシンなどのプロテアーゼ
類、グリコースイソメラーゼ、ウロキナーゼなど
が挙げられる。また、微生物としては、例えばシ
ユードモナス・フルオレツセンス、バチルス・ズ
ブチリス、シユードモナス・エルギノーサなどの
細菌類、アスペルギルス・ニガー、リゾプス・ホ
ルモセンシスなどの糸状菌類、ストレプトミセ
ス・グリセウスなどの放線菌類、酵母菌、かびな
どが挙げられる。 Examples of enzymes include oxidases such as glucose oxidase, amino acid oxidase, cholesterol oxidase, and uricase, uriase, creakininase, glutaminase,
Examples include proteases such as penicillinase, catalase, peroxidase, invertase, mutarotase, amylase, papain, and trypsin, glycose isomerase, and urokinase. Examples of microorganisms include bacteria such as Pseudomonas fluorescens, Bacillus subtilis, and Pseudomonas aeruginosa, filamentous fungi such as Aspergillus niger and Rhizopus hormocensis, actinomycetes such as Streptomyces griseus, yeasts, and molds. Examples include.
本発明に係る生理活性物質固定化膜において
は、トリアミン化合物をスペーサーとして用いる
ことにより、アルデヒド化合物と酵素、微生物な
どの生理活性物質とが穏かに結合されており、か
つトリアミン化合物による主鎖延長によつて生理
活性物質はポリスルホン分子と絡み合つており、
この結果として生理活性物質はポリスルホン膜中
に強固に結合されることになるので、高活性で脱
離し難く、活性安定性の指標である活性残存率の
高い固定膜が形成される。
In the bioactive substance-immobilized membrane according to the present invention, the aldehyde compound and the bioactive substance such as an enzyme or microorganism are gently bonded by using a triamine compound as a spacer, and the main chain is extended by the triamine compound. Due to this, physiologically active substances are entangled with polysulfone molecules,
As a result, the physiologically active substance is firmly bound to the polysulfone membrane, resulting in the formation of an immobilized membrane that is highly active, difficult to desorb, and has a high residual activity rate, which is an indicator of activity stability.
次に、実施例について本発明を説明する。 Next, the present invention will be explained with reference to examples.
実施例
ポリスルホン（日産化学製品ｐ−3500）15重量
部およびジメチルホルムアミド85重量部よりなる
ドープ液10ml中に、50％グルタルアルデヒド水溶
液2.4mlおよび１，８−ジアミノ−４−アミノメ
チルオクタン1.0mlを添加し、この混合液をガラ
ス板上にガラス棒を用いて製膜した。Example Into 10 ml of a dope solution consisting of 15 parts by weight of polysulfone (Nissan Chemical Products p-3500) and 85 parts by weight of dimethylformamide, 2.4 ml of a 50% aqueous glutaraldehyde solution and 1.0 ml of 1,8-diamino-4-aminomethyloctane were added. The mixture was added to form a film on a glass plate using a glass rod.
得られたポリスルホン膜を１cm×１cmの大きさ
に切断し、濃度１mg／mlの枯草菌起源アルカリプ
ロテアーゼ（長瀬産業販売品、酵素番号E.
C.3.4.4.16）の水溶液中に、４℃の条件下に24時
間浸漬し、ポリスルホン膜に上記酵素を固定させ
た。その後、酵素固定膜は、100mlの蒸溜水で洗
浄された。 The obtained polysulfone membrane was cut into pieces of 1 cm x 1 cm and treated with alkaline protease derived from Bacillus subtilis at a concentration of 1 mg/ml (product sold by Nagase Sangyo, enzyme number E.
The enzyme was immobilized on the polysulfone membrane by immersion in an aqueous solution of C.3.4.4.16) at 4°C for 24 hours. The enzyme-immobilized membrane was then washed with 100 ml of distilled water.
この酵素固定膜の活性が、カゼイン基質を用
い、アンソン−萩原氏変法によつて測定された。
その測定条件は、酢酸緩衝液中、PH7.5、温度35
℃、紫外線吸収波数660nmである。また、固定化
酵素を、４℃の酢酸緩衝液５ml中に24時間浸漬し
た後、同様に酵素活性を測定することにより、固
定化酵素の脱離の有無が判定された。更に、固定
化酵素を、４℃の酢酸緩衝液中に30日間浸漬した
後の酵素活性を測定して、残存活性率を算出し
た。得られた結果は、後記表に示される。 The activity of this enzyme-immobilized membrane was measured by a modified Anson-Hagiwara method using a casein substrate.
The measurement conditions are in acetate buffer, pH 7.5, temperature 35
℃, and the ultraviolet absorption wave number is 660 nm. Furthermore, the presence or absence of detachment of the immobilized enzyme was determined by immersing the immobilized enzyme in 5 ml of acetate buffer at 4° C. for 24 hours and then measuring the enzyme activity in the same manner. Furthermore, the enzyme activity was measured after the immobilized enzyme was immersed in an acetate buffer solution at 4° C. for 30 days, and the residual activity rate was calculated. The results obtained are shown in the table below.
比較例
実施例において、１，８−ジアミノ−４−アミ
ノメチルオクタンが用いられなかつた。得られた
酵素固定ポリスルホン膜について、実施例と同様
の測定が行われた。Comparative Example In the example, 1,8-diamino-4-aminomethyloctane was not used. The same measurements as in Examples were performed on the obtained enzyme-immobilized polysulfone membrane.
なお、ポリスルホン膜中の固定化酵素量は、ケ
ルダール窒素分析計（三菱化成製）の測定結果か
ら、上記実施例および比較例の膜共ほぼ同一であ
ることが確認されている。 The amount of immobilized enzyme in the polysulfone membrane was confirmed to be almost the same in the membranes of the above examples and comparative examples, based on the measurement results of a Kjeldahl nitrogen analyzer (manufactured by Mitsubishi Kasei).
表測定項目 実施例 比較例 膜の酵素活性（ｍＵ／cm2） 5.6 4.5 脱離酵素の有無 なし あり 残存活性率（％） 85.3 63.5 Table Measurement Items Examples and Comparative Examples Membrane enzyme activity (mU/cm 2 ) 5.6 4.5 Presence or absence of elimination enzyme None Yes Residual activity rate (%) 85.3 63.5
Claims (1)
物およびトリアミン化合物を添加した混合液を用
いて製膜することを特徴とする生理活性物質固定
化膜の製造法。 ２ アルデヒド化合物がグルタルアルデヒドであ
る特許請求の範囲第１項記載の生理活性物質固定
化膜の製造法。 ３ トリアミン化合物が１，８−ジアミノ−４−
アミノメチルオクタンである特許請求の範囲第１
項記載の生理活性物質固定化膜の製造法。 ４ 酵素の固定化に用いられる特許請求の範囲第
１項記載の生理活性物質固定化膜の製造法。 ５ 微生物の固定化に用いられる特許請求の範囲
第１項記載の生理活性物質固定化膜の製造法。[Scope of Claims] 1. A method for producing a physiologically active substance-immobilized membrane, characterized in that the membrane is formed using a mixed solution in which an aldehyde compound and a triamine compound are added to a polysulfone dope solution. 2. The method for producing a physiologically active substance-immobilized membrane according to claim 1, wherein the aldehyde compound is glutaraldehyde. 3 The triamine compound is 1,8-diamino-4-
Claim 1 which is aminomethyloctane
A method for producing a physiologically active substance-immobilized membrane as described in Section 1. 4. A method for producing a physiologically active substance-immobilized membrane according to claim 1, which is used for enzyme immobilization. 5. A method for producing a physiologically active substance-immobilized membrane according to claim 1, which is used for immobilizing microorganisms.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59178044A JPS6157628A (en) | 1984-08-27 | 1984-08-27 | Production of physiologically active substance immobilized film |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59178044A JPS6157628A (en) | 1984-08-27 | 1984-08-27 | Production of physiologically active substance immobilized film |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6157628A JPS6157628A (en) | 1986-03-24 |
| JPH047377B2 true JPH047377B2 (en) | 1992-02-10 |
Family
ID=16041609
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP59178044A Granted JPS6157628A (en) | 1984-08-27 | 1984-08-27 | Production of physiologically active substance immobilized film |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6157628A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4824870A (en) * | 1987-09-14 | 1989-04-25 | Gelman Sciences, Inc. | Polyaldehyde activated membranes |
| JP2002246891A (en) * | 2001-02-16 | 2002-08-30 | Mitsubishi Electric Corp | Input buffer circuit and semiconductor device |
| CN116120555B (en) * | 2023-04-14 | 2023-06-20 | 富海(东营)新材料科技有限公司 | Preparation method of enzyme immobilized polysulfone and application of polysulfone |
-
1984
- 1984-08-27 JP JP59178044A patent/JPS6157628A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6157628A (en) | 1986-03-24 |
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