JP2001178457A - Method for immobilizing enzyme and immobilized enzyme - Google Patents
Method for immobilizing enzyme and immobilized enzymeInfo
- Publication number
- JP2001178457A JP2001178457A JP36664199A JP36664199A JP2001178457A JP 2001178457 A JP2001178457 A JP 2001178457A JP 36664199 A JP36664199 A JP 36664199A JP 36664199 A JP36664199 A JP 36664199A JP 2001178457 A JP2001178457 A JP 2001178457A
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- Japan
- Prior art keywords
- enzyme
- structural unit
- immobilized
- hrp
- immobilizing
- 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.)
- Pending
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は酵素の固定化方法及
び固定化酵素に関し、更に詳しくは、酵素サイズに比較
して大きな内径を備えた構造安定性を有する構造ユニッ
トに、酵素を高単位にしかも高度に安定した状態で固定
化することができる酵素の固定化方法及び固定化酵素に
関する。TECHNICAL FIELD The present invention relates to a method for immobilizing an enzyme and an immobilized enzyme. More specifically, the present invention relates to a method for immobilizing an enzyme in a structural unit having a large inner diameter compared to the size of the enzyme and having structural stability. In addition, the present invention relates to a method for immobilizing an enzyme which can be immobilized in a highly stable state, and an immobilized enzyme.
【０００２】[0002]
【従来の技術】従来、酵素の固定化方法としては、酵素
を樹脂ビーズ等に直接固定させる方法、酵素を半透明の
ポリマー被膜により被覆するマイクロカプセル法、酵素
蛋白質の表面をポリエチレングリコールや糖脂質で修飾
して安定化させる表面修飾法等が提案されている。しか
しこれらの方法においては、酵素を固定化担体の表面に
露出状態で固定化したり、固定化用の構造ユニットの構
造安定性が欠けていたりするため、酵素の安定性が不足
し勝ちであった。2. Description of the Related Art Conventionally, methods for immobilizing enzymes include a method in which the enzyme is directly immobilized on resin beads, a microcapsule method in which the enzyme is coated with a translucent polymer film, and a method in which the surface of the enzyme protein is polyethylene glycol or glycolipid. A surface modification method for stabilization by modification with a compound has been proposed. However, in these methods, the enzyme is immobilized in an exposed state on the surface of the immobilization carrier, or the structural stability of the immobilization unit is lacking, so that the enzyme stability tends to be insufficient. .
【０００３】又、例えばアクリルアミドやポリビニルア
ルコール、テトラメトキシシラン（ＴＭＯＳ）や有機基
を有するシラン等のゾルないしはコロイド懸濁液を用
い、そのゲル化反応を利用して酵素を固定化するゾルゲ
ル法も、ある程度の酵素安定化効果は得られるが（川上
ら J. Ferment. Bioeng., 84, 240-242, 1998）、十分
とは言えなかった。A sol-gel method is also known in which a sol or colloidal suspension of, for example, acrylamide, polyvinyl alcohol, tetramethoxysilane (TMOS), or a silane having an organic group is used, and the enzyme is immobilized by utilizing the gelation reaction. Although a certain enzyme stabilizing effect can be obtained (Kawakami et al., J. Ferment. Bioeng., 84, 240-242, 1998), it was not sufficient.
【０００４】これらの方法に対し、本件出願人の出願に
係る特願平１１−２７７０２号に開示された、構造安定
性を有する構造ユニットに酵素を固定化する方法、特に
メソポーラスシリカ多孔体を最適例とする無機多孔質材
料の細孔を構造ユニットとして利用する方法によれば、
非常に高い酵素安定化効果が得られることが確認されて
いる。[0004] In contrast to these methods, a method for immobilizing an enzyme on a structural unit having structural stability disclosed in Japanese Patent Application No. 11-27702 filed by the present applicant, in particular, a mesoporous silica porous material is optimally used. According to the method of using the pores of the inorganic porous material as a structural unit as an example,
It has been confirmed that a very high enzyme stabilizing effect can be obtained.
【０００５】[0005]
【発明が解決しようとする課題】ところで、酵素の固定
化は、熱やｐＨ等に対する酵素の安定性の向上を直接の
目的とするが、その際、固定化担体の単位重量当たり、
高単位（高密度）に酵素を固定化したいと言う実用上の
重要な要求もある。そして無機多孔質材料による酵素固
定化方法は、酵素を高単位に固定化すると言う目的から
も好適なものであるが、特に高単位に酵素を固定したい
場合には、酵素サイズに比較して大きな内径を有する構
造ユニットを利用し、単一の構造ユニット当たりの酵素
の固定量を増大させることが有利である。The immobilization of the enzyme is directly aimed at improving the stability of the enzyme against heat, pH, etc.
There is also a practically important requirement to immobilize enzymes in high units (high density). The method of immobilizing an enzyme using an inorganic porous material is also suitable for the purpose of immobilizing the enzyme in high units, but particularly when the enzyme is to be immobilized in high units, it is larger than the enzyme size. It is advantageous to utilize structural units having an inner diameter to increase the amount of enzyme immobilized per single structural unit.
【０００６】しかし、構造ユニットが酵素サイズに比較
して十分に大きい（例えば、構造ユニットの内径が酵素
サイズの１．２倍以上である）場合、恐らくは酵素が構
造ユニット内で十分堅固に固定されないために、必ずし
も固定化酵素の安定性が高くないと言う問題があった。However, if the structural unit is sufficiently large compared to the size of the enzyme (eg, the inner diameter of the structural unit is 1.2 times or more the size of the enzyme), the enzyme is probably not sufficiently firmly fixed in the structural unit. Therefore, there is a problem that the stability of the immobilized enzyme is not always high.
【０００７】そこで本発明は、酵素固定用の構造ユニッ
トが酵素サイズに比較して十分に大きい場合でも酵素を
安定的に固定化できるようにすること、又、このことに
より、内径の大きな構造ユニットを用いた酵素の高単位
固定法の実用性を向上させることを、解決すべき課題と
する。Accordingly, the present invention has been made to enable the enzyme to be stably immobilized even if the structural unit for immobilizing the enzyme is sufficiently large in comparison with the size of the enzyme. An object of the present invention is to improve the practicability of the high-unit immobilization method for enzymes using the method.
【０００８】[0008]
【課題を解決するための手段】（第１発明の構成）上記
課題を解決するための本願第１発明（請求項１に記載の
発明）の構成は、酵素直径の１．２倍以上の内径を備え
構造安定性を有する構造ユニットに酵素を固定化した
後、前記構造ユニットの開口部及び／又は内部空隙にゾ
ルゲル法によるゲル化物質の網状構造を形成して固定化
酵素の安定性を向上させる、酵素の固定化方法である。Means for Solving the Problems (Structure of the First Invention) The structure of the first invention (the invention of claim 1) for solving the above-mentioned problems has an inner diameter of at least 1.2 times the diameter of the enzyme. After immobilizing the enzyme on the structural unit having structural stability, the network structure of the gelled substance is formed in the openings and / or internal voids of the structural unit by the sol-gel method to improve the stability of the immobilized enzyme. This is a method of immobilizing an enzyme.
【０００９】（第２発明の構成）上記課題を解決するた
めの本願第２発明（請求項２に記載の発明）の構成は、
酵素直径の１．２倍以上の内径を備え構造安定性を有す
る構造ユニットに酵素が固定化されており、かつ、該構
造ユニットの開口部及び／又は内部空隙にはゲル化物質
の網状構造が形成されている、固定化酵素である。(Structure of the Second Invention) The structure of the second invention of the present application (the invention according to claim 2) for solving the above problems is as follows.
An enzyme is immobilized on a structural unit having an inner diameter of 1.2 times or more the diameter of the enzyme and having structural stability, and a network structure of a gelling substance is formed in openings and / or internal voids of the structural unit. An immobilized enzyme that has been formed.
【００１０】（第３発明の構成）上記課題を解決するた
めの本願第３発明（請求項３に記載の発明）の構成は、
前記第２発明に係る構造ユニットが、メソポーラスシリ
カ多孔体における細孔である、固定化酵素である。(Structure of Third Invention) The structure of the third invention of the present application (the invention according to claim 3) for solving the above problems is as follows.
The structural unit according to the second invention is an immobilized enzyme, which is a pore in a mesoporous silica porous material.
【００１１】[0011]
【発明の作用・効果】（第１発明及び第２発明の作用・
効果）第１発明においては、酵素直径の１．２倍以上の
内径を備え構造安定性を有する構造ユニットに酵素を固
定化するので、酵素の高単位（高密度）な固定が可能で
ある。Operation and effect of the present invention (operation and effect of the first and second inventions)
Effect) In the first invention, the enzyme is immobilized on a structural unit having an inner diameter of 1.2 times or more the diameter of the enzyme and having structural stability, so that a high unit (high density) immobilization of the enzyme is possible.
【００１２】そして、酵素を固定した構造ユニットにお
いて、その開口部及び／又は内部空隙にゾルゲル法によ
るゲル化物質の網状構造を形成するので、固定化酵素は
この網状構造によって立体構造の大きな変化を拘束さ
れ、比較的大きい構造ユニット中においても十分に堅固
に固定されて安定化する。なお、ゲル化物質の網状構造
は多くのチャネルもしくは隙間を伴うので、酵素活性の
発現に必要な酵素立体構造の若干の変化は許容され、
又、構造ユニットの開口部や内部空間を完全に閉塞する
ことはないので、酵素と基質との接触を阻害しない。In the structural unit in which the enzyme is immobilized, a network structure of the gelled substance is formed by the sol-gel method in the opening and / or the internal space, and the immobilized enzyme undergoes a large change in the three-dimensional structure due to the network structure. It is constrained and is sufficiently firmly fixed and stable even in relatively large structural units. In addition, since the network structure of the gelling substance involves many channels or gaps, slight changes in the enzyme three-dimensional structure required for the expression of the enzyme activity are allowed,
In addition, since the opening and the internal space of the structural unit are not completely closed, the contact between the enzyme and the substrate is not hindered.
【００１３】従って、第１発明の結果として得られた第
２発明に係る固定化酵素は、酵素が高単位に固定化され
ている点、それらの固定化酵素が十分に安定化されてい
る点、及び酵素が基質と容易に接触できる固定化構造で
ある点から、非常に高くかつ安定した酵素活性を発現す
ることができる。Accordingly, the immobilized enzyme according to the second invention obtained as a result of the first invention is characterized in that the enzyme is immobilized in high units and that the immobilized enzyme is sufficiently stabilized. , And an immobilized structure in which the enzyme can easily come into contact with the substrate, so that a very high and stable enzyme activity can be expressed.
【００１４】（第３発明の作用・効果）上記第２発明に
係る固定化酵素において、その構造ユニットが第３発明
のようにメソポーラスシリカ多孔体における細孔である
場合、特に高い酵素安定化効果が得られ、しかも酵素を
高単位に固定すると言う目的からも好適である。(Function / Effect of the Third Invention) In the immobilized enzyme according to the second invention, when the structural unit is a pore in the mesoporous silica porous material as in the third invention, a particularly high enzyme stabilizing effect is obtained. It is also suitable for the purpose of fixing the enzyme to a high unit.
【００１５】[0015]
【発明の実施の形態】次に、第１発明〜第３発明の実施
の形態について説明する。以下において単に「本発明」
と言うときは、第１発明〜第３発明を一括して指してい
る。Next, embodiments of the first to third inventions will be described. In the following, simply "the present invention"
When it says, it points to 1st invention-3rd invention collectively.
【００１６】〔固定化酵素〕本発明に係る固定化酵素
は、酵素直径の１．２倍以上の内径、特に好ましくは
１．５〜３．０倍程度の内径を備えた構造安定性を有す
る構造ユニットの中に酵素が固定され、しかも該構造ユ
ニットの開口部及び／又は内部空隙にはゲル化物質の網
状構造が形成されているものを言う。構造ユニットの内
径が酵素直径より僅かに大きい場合に比較して、一般的
には、例えば酵素直径の１．５倍以上の内径を有する構
造ユニットには１．５倍以上の酵素を固定することがで
きる。酵素は高単位に固定されていることが好ましい
が、高単位に固定されていない場合でも本発明の良好な
酵素安定化効果が得られる。[Immobilized enzyme] The immobilized enzyme according to the present invention has a structural stability having an inner diameter of 1.2 times or more, particularly preferably about 1.5 to 3.0 times, the diameter of the enzyme. An enzyme is immobilized in a structural unit, and a network structure of a gelling substance is formed in openings and / or internal voids of the structural unit. In general, for example, 1.5 times or more of the enzyme is immobilized on a structural unit having an inside diameter of 1.5 times or more the enzyme diameter as compared with the case where the inside diameter of the structural unit is slightly larger than the enzyme diameter. Can be. Although the enzyme is preferably fixed to a high unit, the good enzyme stabilizing effect of the present invention can be obtained even when the enzyme is not fixed to a high unit.
【００１７】本発明において利用可能な酵素の種類は全
く限定されない。又、上記「酵素」とは、通常の酵素蛋
白質分子、又はその活性ユニット（活性部位を含む酵素
の断片）を言う。構造ユニット中には、１種類の酵素だ
けが固定されていても良く、例えば特定の一連の反応に
関わる２種類以上の酵素が同時に固定されていても良
い。後者の場合において、２種類以上の酵素は同一の多
孔体等における別々の構造ユニット中に固定されていて
も良く、同一の構造ユニット中に固定されていても良
い。[0017] The types of enzymes that can be used in the present invention are not at all limited. The “enzyme” refers to a normal enzyme protein molecule or its active unit (enzyme fragment containing an active site). In the structural unit, only one type of enzyme may be immobilized. For example, two or more types of enzymes involved in a specific series of reactions may be immobilized simultaneously. In the latter case, two or more types of enzymes may be fixed in separate structural units in the same porous body or the like, or may be fixed in the same structural unit.
【００１８】〔構造ユニット及び多孔質材料〕構造ユニ
ットとしては、前記の内径を備えた構造安定性を有する
ものが用いられる。構造ユニットは無機材料から構成さ
れても良く、有機ポリマー等の有機材料から構成されて
も良い。そしてアンカーユニット（構造ユニットと酵素
とを連結する水酸基，アミノ基，シラン誘導体等の官能
基等を備えた要素）を備えた構造ユニットが、酵素安定
化作用の強さから特に好ましい。有機材料からなる構造
ユニットにおいては、酵素の周りを、場合によってはア
ンカーユニットを介して被覆するためのポリマー形成反
応が必要である。上記ポリマーやそれを構成するモノマ
ーの種類は、発明の目的を阻害しない限り特段に限定さ
れない。[Structural Unit and Porous Material] As the structural unit, a structural unit having the above inner diameter and having structural stability is used. The structural unit may be made of an inorganic material, or may be made of an organic material such as an organic polymer. A structural unit having an anchor unit (an element having a functional group such as a hydroxyl group, an amino group, or a silane derivative that connects the structural unit to the enzyme) is particularly preferable from the viewpoint of the enzyme stabilizing effect. In structural units made of organic materials, a polymer-forming reaction is required to coat around the enzyme, possibly via an anchor unit. The kind of the polymer and the monomer constituting the polymer are not particularly limited as long as the object of the invention is not hindered.
【００１９】無機材料からなる構造安定性を備えた構造
ユニットは、例えばケイ酸やアルミナ等の各種金属酸化
物、ケイ酸と他種の金属との複合酸化物等によって構成
することができる（無機多孔質材料）。例えば、ケイ酸
からなる構造ユニットの形成方法においては、カネマイ
トのような層状シリケート，アルコキシシラン，シリカ
ゲル，水ガラス，ケイ酸ソーダ等を好ましく用いること
ができる。The structural unit made of an inorganic material and having structural stability can be composed of, for example, various metal oxides such as silicic acid and alumina, and a composite oxide of silicic acid and other metals. Porous material). For example, in a method of forming a structural unit composed of silicic acid, a layered silicate such as kanemite, alkoxysilane, silica gel, water glass, sodium silicate, or the like can be preferably used.
【００２０】無機材料から構造ユニットを作製するに
は、無機材料を界面活性剤（テンプレート物質）と混合
反応させ、界面活性剤のミセルのまわりに無機の骨格が
形成された界面活性剤／無機複合体を形成させた後、例
えば４００°Ｃ〜６００°Ｃでの焼成や、有機溶剤抽出
等により界面活性剤を除去して、界面活性剤のミセルと
同じ形状のメソポア細孔を無機骨格中に形成することが
できる（メソポーラスシリカ多孔体）。In order to prepare a structural unit from an inorganic material, the inorganic material is mixed and reacted with a surfactant (template substance), and a surfactant / inorganic composite in which an inorganic skeleton is formed around micelles of the surfactant. After the body is formed, for example, baking at 400 ° C. to 600 ° C., or removing the surfactant by extracting with an organic solvent, the mesopore pores having the same shape as the micelle of the surfactant are formed in the inorganic skeleton. It can be formed (porous mesoporous silica).
【００２１】上記構造ユニットの作製方法において、ケ
イ素含有化合物例えばケイ酸を出発材料とする場合に
は、カネマイトの如き層状シリケートをまず形成し、こ
の層間にミセルを挿入し、ミセルが存在しない層間をシ
リケート分子で繋ぎ、その後ミセルを除去して細孔を形
成することができる。又、水ガラスのようなケイ素含有
物質を出発材料とし、ミセルの周囲にシリケートモノマ
ーを集合させて重合させることによりシリカを形成し、
次いでミセル分子を取り除いて細孔を形成することもで
きる。この場合、ミセルは通常柱状となり、その結果柱
状の細孔が形成される。In the above method for producing a structural unit, when a silicon-containing compound such as silicic acid is used as a starting material, a layered silicate such as kanemite is first formed, micelles are inserted between the layers, and a layer containing no micelles is formed. The pores can be formed by connecting with silicate molecules and then removing the micelles. In addition, a silicon-containing substance such as water glass is used as a starting material, silica is formed by assembling and polymerizing silicate monomers around micelles,
The micelle molecules can then be removed to form pores. In this case, the micelles are usually columnar, resulting in columnar pores.
【００２２】これらの場合において、界面活性剤のアル
キル鎖の長さを変えてミセルの径を変化させることによ
り、構造ユニットの内径を制御することができる。又、
界面活性剤と併せ、トリメチルベンゼン，トリプロピル
ベンゼン等の比較的疎水性の分子を添加することによ
り、ミセルを膨潤させ、結果的に更に大きな内径の構造
ユニットを形成することもできる。In these cases, the inner diameter of the structural unit can be controlled by changing the length of the micelle by changing the length of the alkyl chain of the surfactant. or,
By adding relatively hydrophobic molecules such as trimethylbenzene and tripropylbenzene in combination with the surfactant, the micelles can be swollen, and as a result, a structural unit having a larger inner diameter can be formed.
【００２３】構造ユニットの形態としては粉末状，顆粒
状，シート状，バルク状，膜状等がある。個々の細孔で
ある構造ユニットの内径は、固定される酵素直径の１．
２倍以上、とりわけ、１．５〜３．０倍程度であること
が好ましい。具体的な内径値は酵素直径との関係で決定
されるので一律には規定できないが、例えば５〜２０ｎ
ｍ程度とすることができる。The form of the structural unit includes powder, granule, sheet, bulk, and film. The internal diameter of the structural unit, which is an individual pore, is 1. 1.
It is preferably at least 2 times, particularly preferably about 1.5 to 3.0 times. Since the specific inner diameter value is determined in relation to the enzyme diameter, it cannot be specified uniformly, but for example, 5 to 20 n
m.
【００２４】〔ゲル化物質の網状構造〕構造ユニットに
固定化された酵素を安定化させるゲル化物質の網状構造
は、酵素の固定化後、ゾルゲル法により形成される。本
発明において「ゾルゲル法」とは、高分子ゲル化物質を
構成するべき低分子原料のゾルないしはコロイド懸濁液
中に固定化酵素を共存させたもとで、前記低分子原料の
ゲル化を行う方法を言う。ゾルゲル法の実施により形成
されたゲル化物質は、構造ユニットの開口部及び／又は
内部空隙に、チャネルや隙間の多い網状構造体として形
成される。[Network Structure of Gelling Substance] The network structure of the gelling substance for stabilizing the enzyme immobilized in the structural unit is formed by the sol-gel method after the immobilization of the enzyme. In the present invention, the "sol-gel method" refers to a method of gelling the low-molecular material under the coexistence of an immobilized enzyme in a sol or a colloidal suspension of the low-molecular material to constitute a high-molecular gelation substance. Say The gelled substance formed by performing the sol-gel method is formed as a network having many channels and gaps in openings and / or internal voids of the structural unit.
【００２５】上記低分子原料の種類は限定されないが、
例えば前記従来技術の項で記載したアクリルアミドやポ
リビニルアルコール、テトラメトキシシラン（ＴＭＯ
Ｓ）や有機基を有するシラン等を用いることができる。
又、本発明に係る前記構造ユニットの構成原料、例えば
ケイ酸やアルミナ等の各種金属酸化物，ケイ酸と他種の
金属との複合酸化物等，アルコキシシラン，シリカゲ
ル，水ガラス，ケイ酸ソーダ等も、一般的に有効に利用
することができる。The type of the low molecular weight raw material is not limited.
For example, acrylamide, polyvinyl alcohol, tetramethoxysilane (TMO)
S) or silane having an organic group can be used.
Also, constituent materials of the structural unit according to the present invention, for example, various metal oxides such as silicic acid and alumina, composite oxides of silicic acid and other metals, alkoxysilane, silica gel, water glass, sodium silicate Etc. can generally be used effectively.
【００２６】ゾルゲル法の実施条件、例えば緩衝溶液の
使用，ゲル化すべきゾルないしはコロイド懸濁液中にお
ける低分子原料の濃度、ｐＨ調整，反応時の加熱もしく
は冷却の要否等の点については、一律には限定されな
い。低分子原料としてアルコキシシランを用いる場合に
おいて、より好ましいゾルゲル法の実施条件として、ジ
メチル−ジメトキシシラン（ＤＭＤＭＯＳ）を添加する
こと、ＤＭＤＭＯＳとテトラメトキシシラン（ＴＭＯ
Ｓ）とを１：１〜１：３の割合に添加すること、ゲル化
の際のｐＨを５〜８に上げること、等の点を挙げること
ができる。The conditions for the sol-gel method, such as the use of a buffer solution, the concentration of low-molecular materials in the sol or colloidal suspension to be gelled, the pH adjustment, and the necessity of heating or cooling during the reaction, are as follows. It is not limited uniformly. When alkoxysilane is used as a low-molecular-weight raw material, more preferable sol-gel method conditions include adding dimethyl-dimethoxysilane (DMDMOS), and adding DMDMOS and tetramethoxysilane (TMO).
And S) in a ratio of 1: 1 to 1: 3, and increasing the pH during gelation to 5 to 8 and the like.
【００２７】[0027]
【発明の有益な実施形態】本発明は、以下の様な有益な
実施形態において、併記する作用・効果を伴って実施す
ることができる。 （１）酵素直径の１．２倍以上の内径を備え構造安定性
を有する構造ユニットに酵素を固定化した後、前記構造
ユニットの開口部及び／又は内部空隙にゾルゲル法によ
るゲル化物質の網状構造を形成して固定化酵素の安定性
を向上させる酵素の固定化方法、及びそのような固定化
酵素。これにより、酵素を高単位にかつ安定的に固定で
きる。 （２）上記構造ユニットの内径が酵素直径の１．５〜
３．０倍程度であり、あるいは５〜２０ｎｍ程度であ
る。これにより、酵素の高単位（高密度）固定と固定化
酵素の安定性とを、特にバランス良く両立させることが
できる。 （３）上記構造ユニットがアンカーユニットを備えてい
る。これにより、構造ユニットの酵素固定化作用が強化
される。 （４）上記構造ユニットが、ケイ酸やアルミナ等の各種
金属酸化物、ケイ酸と他種の金属との複合酸化物等によ
って構成された無機多孔質材料の細孔である。これによ
り、構造ユニットの好ましい構造安定性が得られる。 （５）上記構造ユニットが、メソポーラスシリカ多孔体
の細孔である。これにより、特に優れた酵素固定化作用
が得られ、かつ酵素を高単位に固定化できる。 （６）上記ゾルゲル法に用いる低分子原料が、アクリル
アミドやポリビニルアルコール、テトラメトキシシラン
（ＴＭＯＳ）や有機基を有するシランである。 （７）上記ゾルゲル法に用いる低分子原料が、ケイ酸や
アルミナ等の各種金属酸化物，ケイ酸と他種の金属との
複合酸化物等，アルコキシシラン，シリカゲル，水ガラ
ス，ケイ酸ソーダ等である。 （８）上記ゾルゲル法に用いる低分子原料がアルコキシ
シランである場合において、ゾルゲル法の実施条件とし
て、ＤＭＤＭＯＳを添加すること、ＤＭＤＭＯＳとＴＭ
ＯＳとを１：１〜１：３の割合に添加すること、又はゲ
ル化の際のｐＨを５〜８に上げること。これにより、ゾ
ルゲル法をより効果的に実施することができる。Advantageous Embodiments of the Invention The present invention can be carried out in the following advantageous embodiments with the following actions and effects. (1) After immobilizing an enzyme on a structural unit having an inner diameter of 1.2 times or more the diameter of the enzyme and having structural stability, a network of a gelled substance by a sol-gel method is formed in an opening and / or an internal space of the structural unit. An enzyme immobilization method for forming a structure to improve the stability of the immobilized enzyme, and such an immobilized enzyme. Thereby, the enzyme can be stably immobilized in high units. (2) The inner diameter of the structural unit is 1.5 to the enzyme diameter.
It is about 3.0 times, or about 5 to 20 nm. This makes it possible to achieve both a high unit (high density) immobilization of the enzyme and the stability of the immobilized enzyme in a particularly well-balanced manner. (3) The structural unit has an anchor unit. Thereby, the enzyme immobilizing action of the structural unit is enhanced. (4) The structural unit is a pore of an inorganic porous material composed of various metal oxides such as silicic acid and alumina, and a composite oxide of silicic acid and another metal. Thereby, favorable structural stability of the structural unit is obtained. (5) The structural unit is a pore of a mesoporous silica porous material. Thereby, particularly excellent enzyme immobilizing action can be obtained, and the enzyme can be immobilized in high units. (6) The low-molecular material used in the sol-gel method is acrylamide, polyvinyl alcohol, tetramethoxysilane (TMOS), or silane having an organic group. (7) Low molecular weight raw materials used in the sol-gel method include various metal oxides such as silicic acid and alumina, composite oxides of silicic acid and other metals, alkoxysilane, silica gel, water glass, sodium silicate, etc. It is. (8) When the low-molecular material used in the sol-gel method is an alkoxysilane, the conditions for the sol-gel method include adding DMDMOS, DMDMOS and TM
Adding OS in a ratio of 1: 1 to 1: 3, or increasing the pH during gelation to 5 to 8. Thereby, the sol-gel method can be more effectively performed.
【００２８】[0028]
【実施例】〔実施例１：ＦＳＭ７０の合成〕１００ｍｌ
のビーカーに５．０ｇ（0.028mol）のδ−Ｎａ2Ｓｉ2Ｏ
5（カネマイト）及び５０ｍｌのイオン交換水を入れ、
約２５°Ｃで３時間攪拌してカチオン交換し、次いで水
溶液を濾過してδ−Ｎａ1.6Ｈ0.4Ｓｉ2Ｏ5の沈殿物を得
た。この沈殿物に５０ｍｌのイオン交換水を加え、均一
な分散液になるまで攪拌した。これをＡ液とする。EXAMPLES [ Example 1: Synthesis of FSM70 ] 100 ml
5.0 g (0.028 mol) of δ-Na 2 Si 2 O
5 Add (kanemite) and 50 ml of ion-exchanged water,
Cation exchange was performed by stirring at about 25 ° C. for 3 hours, and the aqueous solution was filtered to obtain a precipitate of δ-Na 1.6 H 0.4 Si 2 O 5 . 50 ml of ion-exchanged water was added to the precipitate, and the mixture was stirred until a uniform dispersion was obtained. This is designated as solution A.
【００２９】１００ｍｌの三角フラスコに４．０ｇ（0.
01 mol）のドコシルトリメチルアンモニウムクロリド
（ＤＴＭＡ−Cl）及び５０ｍｌのイオン交換水を入れて
６０°Ｃで攪拌し、完全透明液になってから、８．０ｇ
（ 0.04mol）のトリイソプロピルベンゼン（ＴＩＰＢ）
を添加して激しく１０分間攪拌した後、６０°Ｃで保持
した。これをＢ液とする。In a 100 ml Erlenmeyer flask, 4.0 g (0.
01 mol) of docosyltrimethylammonium chloride (DTMA-Cl) and 50 ml of ion-exchanged water and stirred at 60 ° C. until a completely clear liquid was obtained.
(0.04 mol) of triisopropylbenzene (TIPB)
Was added and stirred vigorously for 10 minutes, and then kept at 60 ° C. This is designated as solution B.
【００３０】上記Ａ液を２５０ｍｌ容の三口丸形フラス
コに移して激しく攪拌しながら、それにＢ液を徐々に添
加し８０°Ｃまで昇温させ、続けて同温度で３時間恒温
反応させた。次に２Ｎ塩酸を用いて反応液のｐＨを８．
５±０．１まで調整し、続けて３時間攪拌した。反応終
了後すぐに濾過し、２００ｍｌずつのイオン交換水で５
回洗浄濾過した。The solution A was transferred to a 250 ml three-necked round flask, and while vigorously stirring, the solution B was gradually added thereto, and the temperature was raised to 80 ° C., followed by a constant temperature reaction at the same temperature for 3 hours. Next, the pH of the reaction solution was adjusted to 8 using 2N hydrochloric acid.
The mixture was adjusted to 5 ± 0.1 and continuously stirred for 3 hours. Immediately after the completion of the reaction, the mixture was filtered, and 5 ml each of 200 ml of ion-exchanged water.
Washed and filtered twice.
【００３１】生成物を４５°Ｃで２４時間風乾させ、次
いで５５０°Ｃの電気炉で６時間焼成することによりテ
ンプレート物質を除き、白色粉末を約４．５ｇ得た。こ
の白色粉末の構造をＸ線回折装置（理学社製ＲＡＤ−
Ｂ）で確認し、その細孔径や表面積及び細孔総容積をＮ
2吸着装置（Ａｕｔｏｓｏｒｂ）で測定した結果、白色
粉末が前記した構造ユニット（平均細孔径約７０Åの細
孔）を備えるメソポーラスシリカ多孔体の粉末であるこ
とを確認した。これをＦＳＭ７０と呼ぶ。The product was air-dried at 45 ° C. for 24 hours, and then calcined in an electric furnace at 550 ° C. for 6 hours to remove the template substance to obtain about 4.5 g of a white powder. An X-ray diffractometer (RAD-RAD-
B), and confirm the pore diameter, surface area and total pore volume by N
2 As a result of measurement using an adsorption device (Autosorb), it was confirmed that the white powder was a mesoporous silica powder having the above-mentioned structural unit (pores having an average pore diameter of about 70 °). This is called FSM70.
【００３２】〔実施例２：ＦＳＭ９０の合成〕Ｂ液の調
製時に用いるＤＴＭＡ−Clを８．０ｇ（0.02 mol）に変
更した点以外は、全て実施例１と同様の操作を行い、白
色粉末を約４．５ｇ得た。この白色粉末の構造確認、及
び細孔径や表面積及び細孔総容積の測定を行い、白色粉
末が前記した構造ユニット（平均細孔径約９０Åの細
孔）を備えるメソポーラスシリカ多孔体の粉末であるこ
とを確認した。これをＦＳＭ９０と呼ぶ。 Example 2: Synthesis of FSM90 Except that the amount of DTMA-Cl used in the preparation of solution B was changed to 8.0 g (0.02 mol), the same operation as in Example 1 was carried out to obtain a white powder. About 4.5 g was obtained. The structure of the white powder is confirmed, and the pore diameter, surface area and total volume of the pores are measured. The white powder is a mesoporous silica powder having the above-mentioned structural unit (pores having an average pore diameter of about 90 °). It was confirmed. This is called FSM90.
【００３３】〔実施例３：ＦＳＭ７０及びＦＳＭ９０に
よる酵素固定化〕ＦＳＭ７０と、ＦＳＭ９０とのそれぞ
れ２５０ｍｇを、１ｍＬの脱イオン水に酵素ペルオキシ
ダーゼ（ＨＲＰ）を１０ｍｇ溶解させたｐＨ６〜７の酵
素水溶液にそれぞれ加え、回転装置（約１００ｒｐｍ）
にかけて４°Ｃで１６時間緩やかに攪拌した。[ Embodiment 3: For FSM70 and FSM90 ]
Enzyme Immobilization ] 250 mg of FSM70 and 250 mg of FSM90 are added to an enzyme aqueous solution of pH 6 to 7 obtained by dissolving 10 mg of enzyme peroxidase (HRP) in 1 mL of deionized water, and the rotating apparatus (about 100 rpm)
And gently stirred at 4 ° C for 16 hours.
【００３４】この混合液を９０００ｒｐｍで１０分間遠
心分離して固形分を回収し、更に脱イオン水５ｍＬによ
る洗浄と、同上の条件での遠心分離とを３回繰返した。
こうして回収した固形分を風乾させ、ＦＳＭ７０による
固定化酵素と、ＦＳＭ７０による固定化酵素とをそれぞ
れ得た。前者をＦＳＭ７０／ＨＲＰと、後者をＦＳＭ９
０／ＨＲＰと、それぞれ呼ぶ。This mixture was centrifuged at 9000 rpm for 10 minutes to collect a solid, and washing with 5 mL of deionized water and centrifugation under the same conditions were repeated three times.
The solid content thus recovered was air-dried to obtain an enzyme immobilized by FSM70 and an enzyme immobilized by FSM70, respectively. The former is FSM70 / HRP and the latter is FSM9
0 / HRP.
【００３５】なお、この実施例とは別に、メソポーラス
シリカ多孔体の平均細孔径（ｎｍ）と酵素ＨＲＰの吸着
量（ｍｇ酵素／ｇ多孔体）との関係を実験的に調べたと
ころ、平均細孔径３．１ｎｍのメソポーラスシリカ多孔
体における吸着量が２８ｍｇ／ｇ、平均細孔径５．１ｎ
ｍのメソポーラスシリカ多孔体における吸着量が１３２
ｍｇ／ｇ、平均細孔径８．９ｎｍのメソポーラスシリカ
多孔体における吸着量が１８３ｍｇ／ｇであった。Apart from this example, the relationship between the average pore diameter (nm) of the porous mesoporous silica and the amount of enzyme HRP adsorbed (mg enzyme / g porous) was experimentally examined. The amount of adsorption on a porous mesoporous silica having a pore size of 3.1 nm is 28 mg / g, and the average pore size is 5.1 n.
m in the porous mesoporous silica material is 132
mg / g, and the amount of adsorption on a mesoporous silica porous material having an average pore diameter of 8.9 nm was 183 mg / g.
【００３６】〔実施例４：ゾルゲル法によるＨＲＰの固
定化〕テトラメトキシシラン（ＴＭＯＳ）０．６ｍＬ
（8.16 m mol）とジメチル−ジメトキシシラン（ＤＭＤ
ＭＯＳ）０．１８７ｍＬ（2.72 m mol）とを、２０ｍＬ
のサンプル瓶中で４°Ｃで混合した。これを攪拌しなが
ら、０．４ｍＬの脱イオン水と４０μＭの塩酸水溶液１
０μＬとを添加し、アルコキシドが完全に分解された
後、ｐＨ７．５のリン酸緩衝溶液２ｍＬを加えた。 Example 4: HRP solidification by sol-gel method
Stabilization ] Tetramethoxysilane (TMOS) 0.6 mL
(8.16 mmol) and dimethyl-dimethoxysilane (DMD
MOS) 0.187 mL (2.72 mmol) and 20 mL
And mixed at 4 ° C in the sample bottle. While stirring this, 0.4 mL of deionized water and 40 μM hydrochloric acid aqueous solution 1 were added.
After the alkoxide was completely decomposed, 2 mL of a pH 7.5 phosphate buffer solution was added.
【００３７】続いて、別途準備した同上のリン酸緩衝溶
液１ｍＬに２０ｍｇのＨＲＰを溶かした溶液を上記サン
プル瓶に添加し１分間攪拌した後、このサンプル瓶を室
温に戻して自然ゲル化させ、更に２４時間エージングさ
せた後に室温で真空乾燥させ、０．７６ｇの固体を得
た。そしてこの固体に対して脱イオン水２０ｍＬを用い
た洗浄を３回行った後、室温で真空乾燥させて０．５４
ｇのゾルゲル法による固定化酵素を得た。これをＨＲＰ
／Ｇｅｌと呼ぶ。Subsequently, a solution prepared by dissolving 20 mg of HRP in 1 mL of the above-prepared phosphate buffer solution was separately added to the above-mentioned sample bottle and stirred for 1 minute. Then, the sample bottle was returned to room temperature to form a natural gel. After further aging for 24 hours, vacuum drying was performed at room temperature to obtain 0.76 g of a solid. The solid was washed three times with 20 mL of deionized water, and then dried at room temperature under vacuum to obtain 0.54
g of the immobilized enzyme obtained by the sol-gel method. This is HRP
/ Gel.
【００３８】〔実施例５：ＦＳＭ／ＨＲＰのゾルゲル法
による安定化〕ＴＭＯＳ ０．６ｍＬ（8.16 m mol）と
ＤＭＤＭＯＳ ０．１８７ｍＬ（2.72m mol）とを２０
ｍＬのサンプル瓶中で４°Ｃで混合した。これを攪拌し
ながら、０．４ｍＬの脱イオン水と４０μＭの塩酸水溶
液１０μＬとを添加し、アルコキシドが完全に分解され
た後、ｐＨ７．５のリン酸緩衝溶液３ｍＬを加えた。 Example 5: Sol-gel method of FSM / HRP
Of TMOS 0.6 mL (8.16 mmol) and DMDMOS 0.187 mL (2.72 mmol) for 20 minutes.
Mix at 4 ° C. in a mL sample bottle. While stirring, 0.4 mL of deionized water and 10 μL of a 40 μM aqueous hydrochloric acid solution were added, and after the alkoxide was completely decomposed, 3 mL of a pH 7.5 phosphate buffer solution was added.
【００３９】上記内容のサンプル瓶２例を準備し、これ
にそれぞれ５０ｍｇのＦＳＭ７０／ＨＲＰとＦＳＭ９０
／ＨＲＰを添加し、３分間攪拌した後、各サンプル瓶を
室温に戻して自然ゲル化させ、更に２４時間エージング
させた後に真空乾燥させ、それぞれ１．０５ｇのゲルを
得た。そしてこのゲルに対して脱イオン水２０ｍＬを用
いた洗浄を３回行った後、室温で真空乾燥させて０．７
５ｇのゾルゲル法安定化ＦＳＭ／ＨＲＰを得た。これら
をそれぞれ、ＦＳＭ７０／ＨＲＰ／Ｇｅｌ及びＦＳＭ９
０／ＨＲＰ／Ｇｅｌと呼ぶ。Two sample bottles having the above contents were prepared, and 50 mg of FSM70 / HRP and 50 mg of FSM90 were added thereto.
After adding / HRP and stirring for 3 minutes, each sample bottle was returned to room temperature to form a natural gel, aged for 24 hours, and dried under vacuum to obtain 1.05 g of each gel. The gel was washed three times with 20 mL of deionized water, dried at room temperature under vacuum, and washed with 0.7 mL of water.
5 g of sol-gel stabilized FSM / HRP were obtained. These are referred to as FSM70 / HRP / Gel and FSM9, respectively.
Called 0 / HRP / Gel.
【００４０】〔実施例６：各固定化酵素の耐熱安定性試
験〕１．５ｍＬのエッペンドルチューブに、実施例３〜
実施例５に係る各固定化酵素、具体的にはＦＳＭ７０／
ＨＲＰ，ＨＲＰ／Ｇｅｌ及びＦＳＭ７０／ＨＲＰ／Ｇｅ
ｌを、それぞれＨＲＰ換算で０．５ｍｇ相当分秤量して
入れ、５０ｍＭのトリス−ＮａＯＡｃ緩衝溶液（ｐＨ
４．０）を２００μＬ加え、７０°Ｃで１時間熱処理し
た後、１０分間遠心分離した。得られた遠心分離の残渣
を更に５００μＬの５０ｍＭトリス−ＨＣｌ緩衝溶液
（ｐＨ７．５）で２回洗浄し、遠心分離して上清を捨て
た。 Example 6: Test of thermostability of each immobilized enzyme
Experiment ] In a 1.5 mL eppendorf tube, Examples 3 to
Each immobilized enzyme according to Example 5, specifically FSM70 /
HRP, HRP / Gel and FSM70 / HRP / Ge
weighed in an amount equivalent to 0.5 mg in terms of HRP, respectively, and added to a 50 mM Tris-NaOAc buffer solution (pH
4.0) was added, and heat-treated at 70 ° C. for 1 hour, followed by centrifugation for 10 minutes. The obtained centrifugation residue was further washed twice with 500 μL of 50 mM Tris-HCl buffer solution (pH 7.5), centrifuged, and the supernatant was discarded.
【００４１】こうして得た遠心分離の残渣に、５０ｍＭ
トリス−ＨＣｌ緩衝溶液（ｐＨ７．５）２００μＬ、５
０００ｐｐｍのフェノール水溶液４μＬ、３０％の過酸
化水素水１μＬをそれぞれ加え、３７°Ｃで３０分間反
応させた後に１０分間遠心分離した。次にその上清を５
０μＬ取り、５０μＬの１％フェリシアンカリウム／１
Ｍグリシン液（ｐＨ９．６）と混ぜ、そして１００μＬ
の１％４−アミノアンチピリン／１Ｍグリシン液（ｐＨ
９．６）を加え、４９０ｎｍで吸光度を測定して、酵素
活性を算出した。The residue of the centrifugation obtained in this way was
200 μL of Tris-HCl buffer solution (pH 7.5), 5
4 μL of a 000 ppm phenol aqueous solution and 1 μL of a 30% hydrogen peroxide solution were added thereto, and the mixture was reacted at 37 ° C. for 30 minutes and then centrifuged for 10 minutes. Next, the supernatant was
Take 0 μL and 50 μL of 1% potassium ferricyan / 1
Mix with M glycine solution (pH 9.6) and add 100 μL
1% 4-aminoantipyrine / 1M glycine solution (pH
9.6) was added, and the absorbance was measured at 490 nm to calculate the enzyme activity.
【００４２】各固定化酵素における酵素活性の算出は、
上記７０°Ｃでの１時間の熱処理を受けていない固定化
酵素における上記同様の測定値に対する相対的な酵素活
性低下率（％）で算出した。又、比較例として、固定化
していないネイティブなＨＲＰについても、同上の耐熱
安定性試験における酵素活性低下率を算出した。The calculation of the enzymatic activity of each immobilized enzyme is as follows:
The relative decrease in the enzymatic activity (%) with respect to the same measured value in the immobilized enzyme not subjected to the heat treatment at 70 ° C. for 1 hour was calculated. As a comparative example, the enzyme activity reduction rate in the heat stability test was calculated for native HRP not immobilized.
【００４３】その結果、酵素活性低下率は、ネイティブ
なＨＲＰが７６．８％、ＨＲＰ／Ｇｅｌが３０．７％、
ＦＳＭ７０／ＨＲＰが１６．２％、ＦＳＭ７０／ＨＲＰ
／Ｇｅｌが１．８％であった。As a result, the enzyme activity reduction rate was 76.8% for native HRP, 30.7% for HRP / Gel,
16.2% FSM70 / HRP, FSM70 / HRP
/ Gel was 1.8%.
【００４４】〔実施例７：各固定化酵素のトルエン中で
の酸化反応活性〕トルエン２０ｍＬに５０ｍＭとなるよ
うに１，２−ジアミノベンゼンを溶解し、この溶液に
１．１Ｍとなるようにｔ−ブチルヒドロキシペルオキサ
イドをデカン溶液に溶解したものを５ｍＬ加えた。 Example 7: Each immobilized enzyme in toluene
Oxidation reaction activity of 1,2-diaminobenzene was dissolved at 50 mM in 20 mL of toluene, and 5 mL of a solution of t-butylhydroxyperoxide dissolved in a decane solution at 1.1 M was added to this solution. .
【００４５】上記のようにして得た同一の溶液に対し
て、固定化していないネイティブなＨＲＰと、実施例３
〜実施例５に係る各固定化酵素、具体的にはＦＳＭ９０
／ＨＲＰ，ＨＲＰ／Ｇｅｌ及びＦＳＭ９０／ＨＲＰ／Ｇ
ｅｌとを、それぞれＨＲＰ換算で０．５ｍｇ相当分秤量
して加え、酸化反応により生じた１，２−ジニトロベン
ゼンの４７０ｎｍにおける吸光度を測定して、１，２−
ジアミノベンゼンの１，２−ジニトロベンゼンへの変換
率に基づいて、酸化反応活性を算出した。In the same solution obtained as described above, non-immobilized native HRP and Example 3
~ Each immobilized enzyme according to Example 5, specifically FSM90
/ HRP, HRP / Gel and FSM90 / HRP / G
and 0.5 mg in terms of HRP were weighed and added, and the absorbance of 1,2-dinitrobenzene generated by the oxidation reaction at 470 nm was measured.
The oxidation reaction activity was calculated based on the conversion ratio of diaminobenzene to 1,2-dinitrobenzene.
【００４６】各酵素における酸化反応活性の算出は、上
記した１０時間のトルエン浸漬を受けていない各酵素に
おける上記同様の測定値に対する相対的な酸化反応活性
低下率（％）で算出した。The oxidation reaction activity of each enzyme was calculated based on the relative reduction rate (%) of the oxidation reaction activity with respect to the same measured value as above for each enzyme which had not been subjected to the above-mentioned toluene immersion for 10 hours.
【００４７】その結果、上記の変換率は、ネイティブな
ＨＲＰが１０．７％、ＨＲＰ／Ｇｅｌが９５．３％、Ｆ
ＳＭ９０／ＨＲＰが７６．１％、ＦＳＭ９０／ＨＲＰ／
Ｇｅｌが９１．７％であった。As a result, the above conversion rates were 10.7% for native HRP, 95.3% for HRP / Gel, and
76.1% of SM90 / HRP, FSM90 / HRP /
Gel was 91.7%.
【００４８】〔実施例８：各固定化酵素をトルエンに晒
した後の残存酵素活性〕ネイティブなＨＲＰと、実施例
７と同一種類の固定化酵素とについて、それぞれＨＲＰ
換算で０．５ｍｇ相当分をトルエン２０ｍＬにに１５時
間浸漬した。 Example 8: Exposing each immobilized enzyme to toluene
Enzyme activity after the reaction ] Native HRP and the same type of immobilized enzyme as in Example 7 were subjected to HRP
A 0.5 mg equivalent was immersed in 20 mL of toluene for 15 hours.
【００４９】これとは別に、トルエン２０ｍＬに５０ｍ
Ｍとなるように１，２−ジアミノベンゼンを溶解し、こ
の溶液に対して、１．１Ｍとなるようにｔ−ブチルヒド
ロキシペルオキサイドをデカン溶液に溶解したものを５
ｍＬ加えた。Separately, 50 mL of toluene is added to 20 mL of toluene.
M, and 1,2-diaminobenzene were dissolved therein. To this solution, t-butylhydroxyperoxide was dissolved in a decane solution such that 1.1 M was obtained.
mL was added.
【００５０】こうして得た同一の溶液に対して、トルエ
ン１０時間浸漬後の前記所定量のネイティブＨＲＰ及び
各固定化酵素を加え、酸化反応により生じた１，２−ジ
ニトロベンゼンの４７０ｎｍにおける吸光度を測定し
て、１，２−ジアミノベンゼンの１，２−ジニトロベン
ゼンへの変換率（％）を算出することにより、トルエン
浸漬後の酸化反応活性を評価した。To the same solution thus obtained, the predetermined amount of native HRP and each immobilized enzyme after immersion in toluene for 10 hours were added, and the absorbance of 1,2-dinitrobenzene generated by the oxidation reaction at 470 nm was measured. Then, the oxidation reaction activity after immersion in toluene was evaluated by calculating the conversion rate (%) of 1,2-diaminobenzene to 1,2-dinitrobenzene.
【００５１】その結果、上記の酸化反応活性低下率は、
ネイティブなＨＲＰが７６．６％、ＨＲＰ／Ｇｅｌが３
８．５％、ＦＳＭ９０／ＨＲＰが２３．１％、ＦＳＭ９
０／ＨＲＰ／Ｇｅｌが１１．６％であった。As a result, the rate of decrease in the oxidation reaction activity is as follows:
76.6% native HRP, 3 HRP / Gel
8.5%, FSM90 / HRP 23.1%, FSM9
0 / HRP / Gel was 11.6%.
Claims (3)
造安定性を有する構造ユニットに酵素を固定化した後、
前記構造ユニットの開口部及び／又は内部空隙にゾルゲ
ル法によるゲル化物質の網状構造を形成して固定化酵素
の安定性を向上させることを特徴とする酵素の固定化方
法。1. After immobilizing an enzyme on a structural unit having an inner diameter of at least 1.2 times the diameter of the enzyme and having structural stability,
A method for immobilizing an enzyme, comprising forming a network structure of a gelling substance by an sol-gel method in an opening and / or an internal space of the structural unit to improve the stability of the immobilized enzyme.
造安定性を有する構造ユニットに酵素が固定化されてお
り、かつ、該構造ユニットの開口部及び／又は内部空隙
にはゲル化物質の網状構造が形成されていることを特徴
とする固定化酵素。2. An enzyme is immobilized on a structural unit having an inner diameter of at least 1.2 times the diameter of the enzyme and having structural stability, and gelation is formed in an opening and / or an internal space of the structural unit. An immobilized enzyme, wherein a network of substances is formed.
カ多孔体における細孔であることを特徴とする請求項２
に記載の固定化酵素。3. The method according to claim 2, wherein the structural unit is a pore in a mesoporous silica porous material.
4. The immobilized enzyme according to 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP36664199A JP2001178457A (en) | 1999-12-24 | 1999-12-24 | Method for immobilizing enzyme and immobilized enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP36664199A JP2001178457A (en) | 1999-12-24 | 1999-12-24 | Method for immobilizing enzyme and immobilized enzyme |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2001178457A true JP2001178457A (en) | 2001-07-03 |
Family
ID=18487287
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP36664199A Pending JP2001178457A (en) | 1999-12-24 | 1999-12-24 | Method for immobilizing enzyme and immobilized enzyme |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2001178457A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005061961A (en) * | 2003-08-11 | 2005-03-10 | Canon Inc | Porous structure |
| JP2009112242A (en) * | 2007-11-06 | 2009-05-28 | National Institute Of Advanced Industrial & Technology | alpha-AMYLASE-FSM BASED MESO PORE POROUS COMPLEX |
| JP2009153448A (en) * | 2007-12-26 | 2009-07-16 | National Institute Of Advanced Industrial & Technology | Hydrolase complex of silica-based meso porous body-starch |
| US7919263B2 (en) | 2003-08-19 | 2011-04-05 | Canon Kabushiki Kaisha | Organic material-immobiling structure and method for production of the same, and peptide and DNA therefor |
| WO2011155536A1 (en) | 2010-06-09 | 2011-12-15 | 日揮触媒化成株式会社 | Support for protein immobilization, immobilized protein and method for producing same |
| CN101680853B (en) * | 2007-06-15 | 2013-09-18 | 株式会社船井电机新应用技术研究所 | Enzyme electrode and enzyme sensor |
-
1999
- 1999-12-24 JP JP36664199A patent/JP2001178457A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005061961A (en) * | 2003-08-11 | 2005-03-10 | Canon Inc | Porous structure |
| US7919263B2 (en) | 2003-08-19 | 2011-04-05 | Canon Kabushiki Kaisha | Organic material-immobiling structure and method for production of the same, and peptide and DNA therefor |
| CN101680853B (en) * | 2007-06-15 | 2013-09-18 | 株式会社船井电机新应用技术研究所 | Enzyme electrode and enzyme sensor |
| JP2009112242A (en) * | 2007-11-06 | 2009-05-28 | National Institute Of Advanced Industrial & Technology | alpha-AMYLASE-FSM BASED MESO PORE POROUS COMPLEX |
| JP2009153448A (en) * | 2007-12-26 | 2009-07-16 | National Institute Of Advanced Industrial & Technology | Hydrolase complex of silica-based meso porous body-starch |
| WO2011155536A1 (en) | 2010-06-09 | 2011-12-15 | 日揮触媒化成株式会社 | Support for protein immobilization, immobilized protein and method for producing same |
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