JPH0716408B2 - Immobilized bioactive substance and method for producing the same - Google Patents
Immobilized bioactive substance and method for producing the sameInfo
- Publication number
- JPH0716408B2 JPH0716408B2 JP6716289A JP6716289A JPH0716408B2 JP H0716408 B2 JPH0716408 B2 JP H0716408B2 JP 6716289 A JP6716289 A JP 6716289A JP 6716289 A JP6716289 A JP 6716289A JP H0716408 B2 JPH0716408 B2 JP H0716408B2
- Authority
- JP
- Japan
- Prior art keywords
- physiologically active
- active substance
- silk fibroin
- immobilized
- gel layer
- 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
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Description
【発明の詳細な説明】 ［産業上の利用分野］ 本発明はバイオセンサー、バイオリアクターなどに利用
される酵素、微生物などの生理活性物質を固定化した繊
維集合体に関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a fiber assembly on which a physiologically active substance such as an enzyme or a microorganism used in a biosensor, a bioreactor or the like is immobilized.
［従来技術及び問題点］ 近年、医療、化学工業、環境工業、食品などの分野にお
いては、特異な触媒活性を有する酵素、微生物など（以
下「生理活性物質」という）を安定に、操作性よく利用
するために、これらを担体に固定化した、いわゆる固定
化酵素、固定化微生物が種々提案されている。[Prior Art and Problems] In recent years, in the fields of medical care, chemical industry, environmental industry, food, etc., enzymes and microorganisms having unique catalytic activity (hereinafter referred to as “physiologically active substances”) are stably and easily operated. Various types of so-called immobilized enzymes and immobilized microorganisms in which these are immobilized on a carrier have been proposed for use.
これらの内、担体に繊維集合体を用いたものは、他の形
態のものに比べて、単位体積当たりの基質との接触面積
が大きく、しかも、バイオリアクターなどに使用する際
に、シート形状であるため、特別な充填装置などが必要
なく、取り扱いやすいという利点がある。Among them, the one using a fiber assembly as a carrier has a larger contact area with a substrate per unit volume than other forms, and moreover, when used in a bioreactor or the like, it has a sheet shape. Therefore, there is an advantage that a special filling device is not required and it is easy to handle.
この繊維集合体に、酵素、酵母、糸状菌などの生理活性
物質を固定化する方法としては、例えば特開昭60−2246
18号公報に示されるような繊維表面にアミノ基、カルボ
キシル基、フェノール基などの特定の官能基を導入し、
これを生理活性物質の持つ官能基と反応させ、共有結合
させる方法や、特開昭60−120988号公報に示されるよう
な耐熱性繊維からなる繊維集合体に、生理活性物質を混
合したゼラチンなどの天然凝固剤を付着させ、天然凝固
剤を加温凝固させる方法が知られている。Examples of a method for immobilizing a physiologically active substance such as an enzyme, yeast or filamentous fungus on this fiber assembly include, for example, JP-A-60-2246.
Introducing a specific functional group such as an amino group, a carboxyl group, a phenol group on the fiber surface as shown in Japanese Patent No. 18,
A method in which this is reacted with a functional group of a physiologically active substance to form a covalent bond, or gelatin mixed with a physiologically active substance in a fiber assembly composed of heat-resistant fibers as shown in JP-A-60-120988 A method is known in which the natural coagulant is attached and the natural coagulant is heated and coagulated.
しかしながら、前者の方法では、繊維に官能基を導入し
たり、共有結合させたりするための操作が煩雑であり、
しかも、共有結合の反応の際に、生理活性物質の活性が
低下する、いわゆる失活が生じやすいという問題があっ
た。However, in the former method, the operation for introducing a functional group into the fiber or covalently bonding is complicated,
Moreover, there has been a problem that the activity of the physiologically active substance is lowered, that is, so-called deactivation is likely to occur during the covalent bond reaction.
一方、後者の方法では、繊維と生理活性物質との間には
実質的に結合はなく、天然凝固剤に包囲される形で生理
活性物質は繊維に固定化されるから、前者の場合のよう
な問題は生じない。しかし、包囲する天然凝固剤が、反
応させるべき基質が生理活性物質の活性中心に移動する
のを阻害して、活性を低下させる場合があり、また、加
温凝固を必要とするため熱に強い生理活性物質しか利用
できないという欠点があった。On the other hand, in the latter method, there is substantially no bond between the fiber and the physiologically active substance, and the physiologically active substance is immobilized on the fiber in a form surrounded by the natural coagulant. Problem does not occur. However, the surrounding natural coagulant may impede the movement of the substrate to be reacted to the active center of the physiologically active substance, resulting in a decrease in activity. Also, it requires warming coagulation and is resistant to heat. It has a drawback that only physiologically active substances can be used.
［発明が解決すべき課題］ 本発明は上記従来技術の問題点を解決すべくなされたも
のであり、生理活性物質を活性の高い状態で、かつ基質
との接触面積の大きい状態で固定化した担体を提供する
ことを目的とする。[Problems to be Solved by the Invention] The present invention has been made to solve the above-mentioned problems of the prior art, in which a physiologically active substance is immobilized in a highly active state and in a large contact area with a substrate. The purpose is to provide a carrier.
［課題を解決する手段］ 本発明は親水性繊維を主体とする繊維集合体の構成繊維
の表面に、タンパク質などの大きな分子は通過できない
が、反応基質などの小さな分子は通過できる絹フィブロ
インのゲル層が形成されており、該ゲル層に生理活性物
質が内包されていることを特徴とする固定化生理活性物
質に関する。[Means for Solving the Problems] The present invention is a silk fibroin gel that can pass large molecules such as proteins but cannot pass small molecules such as reaction substrates on the surface of the constituent fibers of a fiber assembly mainly composed of hydrophilic fibers. The present invention relates to an immobilized physiologically active substance, wherein a layer is formed, and the gel layer contains a physiologically active substance.
また、本発明は親水性繊維を主体とする繊維集合体に、
生理活性物質を含む絹フィブロイン水溶液を含浸する工
程と、これを乾燥する工程と、アルコールにより不溶化
する工程とからなる固定化生理活性物質の製造方法に関
する。Further, the present invention is a fiber assembly mainly composed of hydrophilic fibers,
The present invention relates to a method for producing an immobilized physiologically active substance, which comprises a step of impregnating an aqueous silk fibroin solution containing a physiologically active substance, a step of drying the same, and a step of insolubilizing with an alcohol.
［作用］ 本発明の固定化生理活性物質は、親水性繊維を主体とす
る繊維集合体の表面に、絹フィブロインのゲル層を形成
しているため、繊維とゲル層との親和性が高く、ゲル層
は繊維表面にしっかりと付着する。[Function] Since the immobilized physiologically active substance of the present invention forms a silk fibroin gel layer on the surface of a fiber assembly mainly composed of hydrophilic fibers, the affinity between the fiber and the gel layer is high, The gel layer adheres firmly to the fiber surface.
絹フィブロインのゲル層は、表面部では結晶構造領域が
集中し、内部ではこの領域が少ない不均一構造になって
いると推定される。このため、酵素などの生理活性物質
は分子が大きいのでゲル層からでることはできないが、
低分子である基質や、反応生成物は自由に出入りでき
る。また、ゲル層内では生理活性物質は自由度が大きい
ので、基質などと反応しやすい状態となっている。故
に、生理活性物質は非常に活性が高く、基質と接触しや
すい状態で、繊維表面に固定化されている。It is presumed that the gel layer of silk fibroin has a heterogeneous structure in which the crystal structure region is concentrated on the surface portion and this region is small inside. For this reason, physiologically active substances such as enzymes cannot get out of the gel layer because they have large molecules,
Substrates that are small molecules and reaction products can freely enter and exit. In addition, since the physiologically active substance has a high degree of freedom in the gel layer, it is in a state of easily reacting with a substrate or the like. Therefore, the physiologically active substance has a very high activity and is immobilized on the fiber surface in a state where it is easily contacted with the substrate.
なお、繊維集合体に繊維が三次元的に絡合した不織布を
用いると、基質と接触できる繊維表面の面積が増加して
反応の効率をあげることができ、しかも、基質の通過抵
抗を低くすることができる。When a non-woven fabric in which fibers are entangled three-dimensionally is used in the fiber assembly, the area of the fiber surface that can come into contact with the substrate is increased, and the reaction efficiency can be increased, and further, the passage resistance of the substrate is lowered. be able to.
本発明の製造方法では、親水性繊維を主体とする繊維集
合体に生理活性物質を含む絹フィブロイン水溶液を含浸
するため、絹フィブロイン水溶液を効率よく、しかも均
一に繊維表面に付着することができる。また、この付着
した絹フィブロイン水溶液はこの後、乾燥し、アルコー
ルにより不溶化されるが、とくにアルコールを用いる方
法は短時間の不溶化を可能にする。In the production method of the present invention, since the silk fibroin aqueous solution containing the physiologically active substance is impregnated in the fiber assembly mainly composed of hydrophilic fibers, the silk fibroin aqueous solution can be efficiently and uniformly attached to the fiber surface. Further, the attached silk fibroin aqueous solution is then dried and insolubilized with alcohol. In particular, the method using alcohol enables insolubilization in a short time.
［実施例］ 以下、実施例に基づいて本発明を更に詳細に説明する。[Examples] Hereinafter, the present invention will be described in more detail based on Examples.
本発明に用いられる生理活性物質とは、例えば、グルコ
ースオキシターゼ、アルカリ性フォスファターゼ、アス
パルターゼ、アミラーゼ、インベルターゼなどの酵素、
ニコチンアミドアデニンジヌクレオチド、アデノシント
リホスフェート、補酵素−Ａなどの補酵素、ムコール、
リゾプスなどの糸状菌、サッカロミセス、ピヒア、ハン
ゼヌラなどの酵母などであり、生体触媒活性を有する物
質が用いられる。The physiologically active substance used in the present invention, for example, glucose oxidase, alkaline phosphatase, aspartase, amylase, enzymes such as invertase,
Nicotinamide adenine dinucleotide, adenosine triphosphate, coenzymes such as coenzyme-A, mucor,
Filamentous fungi such as Rhizopus, yeasts such as Saccharomyces, Pichia, Hansenula and the like, and substances having biocatalytic activity are used.
また、本発明に使用される繊維集合体には、不織布、フ
ェルト、織物、編み物、またはこれらの複合体が適して
いる。これらのうち、繊維が三次元的に絡合された不織
布は、基質の通過に対する抵抗が低く、基質と繊維表面
との接触面積が増大するので好ましく、とくに高速柱状
水流により絡合（水流絡合）されたものは、繊維以外の
界面活性剤、接着剤などの余分な成分を実質的に含まな
いため望ましい。Further, as the fiber assembly used in the present invention, non-woven fabric, felt, woven fabric, knitted fabric, or a composite thereof is suitable. Of these, a non-woven fabric in which fibers are three-dimensionally entangled is preferred because it has low resistance to passage of the substrate and increases the contact area between the substrate and the fiber surface, and is particularly entangled by a high-speed columnar water flow (water entanglement). ) Is desirable because it does not substantially contain extra components such as surfactants and adhesives other than fibers.
上記繊維集合体は親水性繊維を主体として構成される。
親水性繊維の量は少なくとも繊維集合体の50重量％は必
要であり、好ましくは70重量％以上、更に好ましくは10
0重量％であるのがよい。The fiber assembly is mainly composed of hydrophilic fibers.
The amount of hydrophilic fibers is required to be at least 50% by weight of the fiber aggregate, preferably 70% by weight or more, more preferably 10% by weight.
It should be 0% by weight.
この親水性繊維としては、例えば、絹繊維、レーヨン繊
維などがとくに好適に用いられる。As the hydrophilic fiber, for example, silk fiber, rayon fiber or the like is particularly preferably used.
繊維集合体の密度は0.04〜0.25g/cm3の範囲にあるのが
よく、0.04g/cm3未満ではゲルの担持量が少なくなるた
め生理活性物質による活性が低下し、一方、0.25g/cm3
を越えると基質の通過抵抗が大きくなってしまう。Density of the fiber aggregate may in the range of 0.04~0.25g / cm 3, is less than 0.04 g / cm 3 was reduced activity due to physiologically active substances for the supported amount of the gel is reduced, whereas, 0.25 g / cm 3
If it exceeds, the passage resistance of the substrate increases.
また、繊維集合体の厚さは0.1〜3.0mmの範囲にあるのが
よく、0.1mm未満では繊維集合体の機械的強度が低いた
め実用に耐えず、3mmを超えると繊維集合体への生理活
性物質含有溶液の均一な含浸が極めて困難となる。Further, the thickness of the fiber aggregate is preferably in the range of 0.1 to 3.0 mm.If it is less than 0.1 mm, the mechanical strength of the fiber aggregate is low and it cannot be used practically. Uniform impregnation of the active substance-containing solution becomes extremely difficult.
上記親水性繊維を主体とする繊維集合体の構成繊維の表
面に、生理活性物質を内包する絹フィブロインのゲル層
が形成される。このゲル層の形成は例えば次の手順で行
なわれる。A gel layer of silk fibroin containing a physiologically active substance is formed on the surface of the constituent fibers of the fiber assembly mainly composed of the hydrophilic fibers. The gel layer is formed, for example, by the following procedure.
まず、絹フィブロイン水溶液を調整し、これに生理活性
物質を混合する。次に、繊維集合体にこの混合液を含浸
して、繊維集合体に含まれる親水性繊維の絹フィブロイ
ン水溶液とのなじみのよさを利用して、構成繊維の表面
に混合液を付着させる。この際、絹フィブロイン水溶液
をより均一に付着させ、かつ繊維集合体内に良好な空隙
を確保させるために、ローラーなどを用いて圧搾しても
よい。この後、室温で風乾し、水を一部除去する。最後
にメタノールなどのアルコールに浸漬して絹フィブロイ
ンを不溶化し、ゲル層を形成する。First, an aqueous silk fibroin solution is prepared, and a physiologically active substance is mixed therein. Next, the fiber assembly is impregnated with this mixed solution, and the mixed solution is attached to the surface of the constituent fibers by utilizing the compatibility of the hydrophilic fibers contained in the fiber assembly with the aqueous silk fibroin solution. At this time, in order to adhere the silk fibroin aqueous solution more uniformly and to secure good voids in the fiber assembly, pressing may be performed using a roller or the like. Then, it is air-dried at room temperature to partially remove water. Finally, it is dipped in alcohol such as methanol to insolubilize the silk fibroin to form a gel layer.
なお、絹フィブロインの不溶化はアルコールによる方法
に限らず、場合によっては、中性塩や希薄酸を用いる方
法や、絹フィブロインに延伸等の応力を加える方法によ
り不溶化してもよい。The method for insolubilizing silk fibroin is not limited to the method using alcohol, but in some cases, it may be insolubilized by a method using a neutral salt or a dilute acid, or a method of applying stress such as stretching to silk fibroin.
この様にして繊維表面に形成された絹フィブロインのゲ
ル層は、結晶領域が表面部へ集中し、ゲル層内部は結晶
領域が少ない、厚み方向に不均一な構造となる。このた
め、ゲル層に内包された生理活性物質は、ゲル層内部で
は比較的自由度の大きな状態で存在するが、表面部を通
過して外部へ出ることはできず、ゲル層内にとじ込めら
れて繊維表面に固定化される。The silk fibroin gel layer thus formed on the fiber surface has a structure in which crystal regions are concentrated on the surface portion, and the inside of the gel layer has few crystal regions and has a non-uniform structure in the thickness direction. For this reason, the physiologically active substance encapsulated in the gel layer exists in a state with a relatively large degree of freedom inside the gel layer, but cannot pass through the surface portion to the outside and is confined in the gel layer. And fixed on the fiber surface.
一方、生理活性物質を用いて処理される反応基質は低分
子であるので、結晶領域の集中したゲル層の表面部も通
過できるため、ゲル層内に侵入し、生理活性物質の活性
中心に到達し、触媒作用を受けて目的とする反応物を生
成する。得られる生成物も低分子であるため、ゲル層の
内部から外部へと移動できる。On the other hand, since the reaction substrate treated with a physiologically active substance is a low molecular weight compound, it can pass through the surface of the gel layer where the crystal region is concentrated, so it penetrates into the gel layer and reaches the active center of the physiologically active substance. Then, it is subjected to a catalytic action to produce a desired reaction product. The resulting product is also a low molecular weight compound and can migrate from the inside to the outside of the gel layer.
以上のような繊維集合体に固定化された生理活性物質
は、フィルム状やビーズ状のゲルに固定化されたものに
比べて比活性が高く、かつ機械的な損傷を受けにくく、
安定性に優れている。The physiologically active substance immobilized on the fiber aggregate as described above has a higher specific activity than that immobilized on a film-like or bead-like gel, and is less likely to be mechanically damaged,
It has excellent stability.
（実施例１） 長さ50mmにカットした家蚕精練絹よりなる繊維ウェブを
水流絡合法により、厚さ0.25mm、見かけ密度0.2g/cm3の
不織布に成形した。別に、家蚕精練絹より得た絹フィブ
ロインの水溶液（2.4％）を調整し、グルコースオキシ
ダーゼ（GOD）（EC.1.1.3.4.Aspergillus niger）を絹
フィブロインに対して２％の割合で加え静かに溶解させ
た。この溶液を不織布に塗布し、マングルにて圧搾し
た。絹フィブロイン溶液の不織布への付着量は、不織布
重量に対して約1.5倍であった。本品を20℃、50％相対
湿度で風乾後、80％メタノール水溶液中に30秒間浸漬し
て絹フィブロインを不溶化させ、酵素固定化織布を得
た。(Example 1) A fibrous web made of refined silkworm domestic silkworm cut to a length of 50 mm was formed into a nonwoven fabric having a thickness of 0.25 mm and an apparent density of 0.2 g / cm 3 by a hydroentangling method. Separately, prepare an aqueous silk fibroin solution (2.4%) obtained from domestic silkworm refined silk, and add glucose oxidase (GOD) (EC.1.1.3.4.Aspergillus niger) at a ratio of 2% to silk fibroin and gently dissolve it. Let This solution was applied to a non-woven fabric and pressed with a mangle. The amount of the silk fibroin solution attached to the nonwoven fabric was about 1.5 times the weight of the nonwoven fabric. This product was air-dried at 20 ° C. and 50% relative humidity, and then immersed in an 80% methanol aqueous solution for 30 seconds to insolubilize silk fibroin to obtain an enzyme-immobilized woven fabric.
固定化GODのグルコース酸化反応における酵素活性を0.1
Mリン酸緩衝液中で測定したところ、第１表に示すよう
に、25℃、pH7の条件で比活性が11.7U/mg遊離酵素に対
する活性収率が9.5％の値が得られた。Enzyme activity of glucose Oxidation of immobilized GOD was 0.1
When measured in M phosphate buffer, a specific activity of 11.7 U / mg free enzyme was obtained at a yield of 9.5% at 25 ° C. and pH 7 as shown in Table 1.
固定化酵素の安定性を検討した結果、60℃以上では遊離
酵素よりもむしろ高い活性を維持しており、熱安定性に
極めて優れていることがわかった。また、至適pHは遊離
酵素が5.5付近であるのに対して、７付近にあり、遊離
酵素よりも高pH側へ移動した。また、水中への絹フィブ
ロインと酵素の溶出度をローリー法及びUV法により測定
したところ、40日後においても絹フィブロインの溶出度
は0.1％以下であり、酵素の溶出は検出されず、極めて
良好であった。As a result of studying the stability of the immobilized enzyme, it was found that the activity was higher than that of the free enzyme at 60 ° C. or higher, and that the thermal stability was extremely excellent. Further, the optimum pH was around 7 while the free enzyme was around 5.5, and it moved to a higher pH side than the free enzyme. Further, when the elution degree of silk fibroin and the enzyme in water was measured by the Lowry method and the UV method, the elution degree of silk fibroin was 0.1% or less even after 40 days, and the elution of the enzyme was not detected and was extremely good. there were.
絹フィブロインの付着状態を走査型電子顕微鏡で観察し
たところ、繊維表面に絹フィブロインがまんべんなく付
着しており、かつ不織布の繊維間の空隙は絹フィブロイ
ンに塞がれることなく残っていた。Observation of the adhering state of silk fibroin with a scanning electron microscope revealed that silk fibroin was evenly adhered to the fiber surface, and voids between fibers of the non-woven fabric remained without being blocked by silk fibroin.
更に、上記絹フィブロイン水溶液の濃度及び酵素配合量
の影響を調べた結果、絹フィブロイン水溶液の濃度１〜
３％の範囲で活性収率が変化せず、一方絹フィブロイン
に対する酵素の添加割合を２％から0.002％と減らすに
つれて活性収率が増加した。酵素添加割合が0.002％の
とき、活性収率は94.2％であり、ほとんど遊離酵素に近
い値が得られた。Furthermore, as a result of investigating the effects of the concentration of the silk fibroin aqueous solution and the amount of the enzyme compounded, the concentration of the silk fibroin aqueous solution was 1 to
The activity yield did not change in the range of 3%, while the activity yield increased as the addition ratio of enzyme to silk fibroin was reduced from 2% to 0.002%. When the enzyme addition rate was 0.002%, the activity yield was 94.2%, which was close to that of the free enzyme.
（実施例２） ビスコースレーヨン（繊度1.5デニール、長さ38mm）の
ウェブを水流絡合法により厚さ0.3mm、見かけ密度0.2g/
cm3の不織布に成形した。実施例１と同様の操作によりG
ODをレーヨン不織布に固定化して酵素活性を測定し、そ
の結果を第１表に示した。(Example 2) A viscose rayon (fineness: 1.5 denier, length: 38 mm) web was formed by a hydroentangling method to have a thickness of 0.3 mm and an apparent density of 0.2 g /
It was formed into a non-woven fabric of cm 3 . By the same operation as in Example 1, G
The OD was immobilized on a rayon nonwoven fabric and the enzyme activity was measured. The results are shown in Table 1.
また、絹フィブロインの付着状態を走査型電子顕微鏡で
観察したところ、繊維表面への絹フィブロインの付着は
均一であり、かつ不織布の繊維間の空隙は絹フィブロイ
ンに塞がれることなく充分残されていた。Further, when the adhesion state of silk fibroin was observed with a scanning electron microscope, the adhesion of silk fibroin to the fiber surface was uniform, and the voids between the fibers of the non-woven fabric were sufficiently left without being blocked by silk fibroin. It was
（比較例１） 繊維をポリエステル繊維（繊度1.5デニール、長さ38m
m）に変えたこと以外は実施例１と全く同様にして酵素
固定化不織布を得た。実施例１と同様の操作によりGOD
をレーヨン不織布に固定化して酵素活性を測定し、実施
例１、２と合わせて第１表に示した。(Comparative Example 1) The fibers are polyester fibers (fineness: 1.5 denier, length: 38 m)
An enzyme-immobilized nonwoven fabric was obtained in exactly the same manner as in Example 1 except that m) was changed. By the same operation as in Example 1, GOD
Was immobilized on a rayon nonwoven fabric and the enzyme activity was measured. The results are shown in Table 1 together with Examples 1 and 2.
第１表から明らかなように、繊維に親水性繊維を用いた
実施例１、２の酵素固定化不織布の比活性は高かった
が、疎水性繊維であるポリエステル繊維を用いた比較例
１では比活性が劣っていた。 As is clear from Table 1, the specific activities of the enzyme-immobilized nonwoven fabrics of Examples 1 and 2 in which hydrophilic fibers were used as fibers were high, but in Comparative Example 1 in which the polyester fibers which are hydrophobic fibers were used, The activity was inferior.
また、走査型電子顕微鏡で観察した結果、絹フィブロイ
ンのポリエステル繊維への付着は絹やレーヨンの場合と
比べて不均一でその付着量も著しく少なかった。As a result of observing with a scanning electron microscope, the adhesion of silk fibroin to the polyester fiber was non-uniform and the adhesion amount was significantly smaller than that of silk or rayon.
（比較例２） 実施例１で用いたGODを含む絹フィブロイン水溶液を、
ポリエチレンフィルム上にキャストして風乾した後、80
％メタノール水溶液中に30秒間浸漬して厚さ20μｍの固
定化酵素フィルムを作成した。(Comparative Example 2) The silk fibroin aqueous solution containing GOD used in Example 1 was
80 after casting on polyethylene film and air-drying
% Aqueous methanol solution for 30 seconds to prepare an immobilized enzyme film having a thickness of 20 μm.
この固定化酵素フィルムの酵素活性を測定したところ、
GODの比活性が3.1U/mg、活性収率が2.6％であり、実施
例１の不織布に固定化した場合に比べて活性がかなり低
かった。When the enzyme activity of this immobilized enzyme film was measured,
The specific activity of GOD was 3.1 U / mg and the activity yield was 2.6%, which was considerably lower than the activity when immobilized on the nonwoven fabric of Example 1.
（比較例３〜４） 酵素の固定化剤として、絹フィブロインをアルギン酸ナ
トリウム又はκ−カラギーナンに代え、アルギン酸ナト
リウムの不溶化は２％塩化カルシウム水溶液で、κ−カ
ラギーナンの不溶化は１％水酸化カリウム水溶液で行な
ったこと以外は、実施例２と同様にして酵素固定化不織
布を得た。(Comparative Examples 3 to 4) As an enzyme immobilizing agent, the silk fibroin was replaced with sodium alginate or κ-carrageenan, and sodium alginate was insolubilized with a 2% calcium chloride aqueous solution, and κ-carrageenan was insolubilized with a 1% potassium hydroxide aqueous solution. An enzyme-immobilized non-woven fabric was obtained in the same manner as in Example 2 except that the above procedure was performed.
この酵素固定化不織布の酵素活性を測定し、実施例２の
結果とともに第２表に示した。The enzyme activity of this enzyme-immobilized nonwoven fabric was measured and is shown in Table 2 together with the results of Example 2.
第２表から明らかなように、絹フィブロインを用いた場
合、アルギン酸ナトリウムやκ−カラギーナンのゲルを
固定化剤に用いた場合に比べて活性収率がかなり優れて
いた。 As is clear from Table 2, the activity yield was significantly better when silk fibroin was used than when sodium alginate or κ-carrageenan gel was used as the immobilizing agent.
また、走査型電子顕微鏡で観察したところ、アルギン酸
ナトリウムやκ−カラギーナンのゲルはレーヨン表面に
均一付着していなかった。Further, when observed with a scanning electron microscope, the gels of sodium alginate and κ-carrageenan were not uniformly attached to the rayon surface.
［発明の効果］ 本発明の固定化生理活性物質は上述のような構成からな
るため、以下に示す効果を奏する。[Effects of the Invention] Since the immobilized physiologically active substance of the present invention has the above-mentioned constitution, it has the following effects.
繊維集合体の構成繊維の表面に生理活性物質を内包す
る絹フィブロインのゲル層が形成されているため、反応
に有効なゲル層の表面積が大きく、固定化生理活性物質
の比活性が高い。Since the silk fibroin gel layer encapsulating the physiologically active substance is formed on the surface of the constituent fibers of the fiber assembly, the surface area of the gel layer effective for the reaction is large and the specific activity of the immobilized physiologically active substance is high.
生理活性物質の固定化が穏やかな条件下で行なえるた
め、担体結合法や天然凝固剤などを用いる場合のような
失活のおそれがない。Since the physiologically active substance can be immobilized under mild conditions, there is no risk of deactivation as in the case of using a carrier binding method or a natural coagulant.
絹フィブロインのゲル層は厚み方向に不均一構造をな
すため、ゲル層内の生理活性物質は自由度が大きく、遊
離状態と変らない活性を有し、しかも、ゲル層からの生
理活性物質の溶出はほとんどない。Since the gel layer of silk fibroin has a non-uniform structure in the thickness direction, the physiologically active substance in the gel layer has a large degree of freedom, has an activity that does not change to a free state, and elutes the physiologically active substance from the gel layer. Almost never.
ゲル層は繊維集合体に固定化されるため、機械的劣化
を受けにくい。Since the gel layer is fixed to the fiber assembly, it is less susceptible to mechanical deterioration.
とくに、繊維集合体に三次元的に絡合した不織布を用
いると、単位体積当たりの反応に有効なゲル層の表面積
が大きくなり、しかも、基質の通過に対する抵抗が低く
なる。In particular, when a non-woven fabric three-dimensionally entangled with a fiber assembly is used, the surface area of the gel layer effective for the reaction per unit volume increases, and the resistance to passage of the substrate decreases.
ゲル層を繊維表面にコーティングすればよいので、親
水性繊維を主体とする繊維集合体であれば、どの様な形
態のものであっても、繊維種のものであっても担体に利
用できる。Since the surface of the fiber may be coated with the gel layer, any form and fiber type can be used as the carrier as long as it is a fiber aggregate mainly composed of hydrophilic fibers.
以上のように、本発明の固定化生理活性物質は優れた効
果を持つため、バイオリアクターやバイオセンターとし
て利用するのに極めて有用である。As described above, since the immobilized physiologically active substance of the present invention has excellent effects, it is extremely useful for use as a bioreactor or biocenter.
Claims (3)
繊維の表面に、タンパク質などの大きな分子は通過でき
ないが、反応基質などの小さな分子は通過できる絹フィ
ブロインのゲル層が形成されており、該ゲル層に生理活
性物質が内包されていることを特徴とする固定化生理活
性物質。1. A silk fibroin gel layer is formed on the surface of constituent fibers of a fiber assembly mainly composed of hydrophilic fibers, in which large molecules such as proteins cannot pass but small molecules such as reaction substrates can pass. And a physiologically active substance is encapsulated in the gel layer.
織布である請求項１に記載の固定化生理活性物質。2. The immobilized physiologically active substance according to claim 1, wherein the fiber assembly is a non-woven fabric in which fibers are entangled three-dimensionally.
理活性物質を含む絹フィブロイン水溶液を含浸する工程
と、これを乾燥する工程と、アルコールにより不溶化す
る工程とからなる固定化生理活性物質の製造方法。3. An immobilized physiological activity comprising a step of impregnating a fiber assembly mainly composed of hydrophilic fibers with an aqueous silk fibroin solution containing a physiologically active substance, a step of drying the same, and a step of insolubilizing it with alcohol. Method of manufacturing substance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6716289A JPH0716408B2 (en) | 1989-03-17 | 1989-03-17 | Immobilized bioactive substance and method for producing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6716289A JPH0716408B2 (en) | 1989-03-17 | 1989-03-17 | Immobilized bioactive substance and method for producing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02245189A JPH02245189A (en) | 1990-09-28 |
| JPH0716408B2 true JPH0716408B2 (en) | 1995-03-01 |
Family
ID=13336920
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6716289A Expired - Lifetime JPH0716408B2 (en) | 1989-03-17 | 1989-03-17 | Immobilized bioactive substance and method for producing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0716408B2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3035834B2 (en) * | 1991-04-11 | 2000-04-24 | 株式会社林原生物化学研究所 | Propolis component-containing solid, its production method and use |
| JP2842980B2 (en) * | 1993-06-02 | 1999-01-06 | 出光石油化学株式会社 | Production method of ultrafine silk fibroin powder |
| US20110189292A1 (en) | 2009-04-20 | 2011-08-04 | Allergan, Inc. | Dermal fillers comprising silk fibroin hydrogels and uses thereof |
| US20110008437A1 (en) | 2009-04-20 | 2011-01-13 | Altman Gregory H | Silk Fibroin Hydrogels and Uses Thereof |
-
1989
- 1989-03-17 JP JP6716289A patent/JPH0716408B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH02245189A (en) | 1990-09-28 |
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