JP5697129B2 - Enzyme activity measurement substrate using FRET and method for producing the same - Google Patents
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
Description
本発明は、酵素の基質及びその製造方法に関する。より具体的には、ＦＲＥＴを利用した酵素の基質及びその製造方法に関する。 The present invention relates to an enzyme substrate and a method for producing the same. More specifically, the present invention relates to an enzyme substrate using FRET and a method for producing the same.
蛍光共鳴エネルギー移動（FRET; Fluorescence Resonance Energy Transfer）を誘導する分子は、ストライヤーらにより分子定規として初めて合成され、以来、様々な分野においてその効果が利用されている。ストライヤーらによって合成された分子は、１００Å以内の鎖長となるペプチドの両末端に蛍光ドナーと蛍光アクセプター導入したもので、ドナーからの蛍光をアクセプターが吸収し、ドナーとは異なる波長の蛍光を発するものである。このペプチドが鎖内で切断されると、ドナーからの蛍光をアクセプターが吸収できなくなるため、アクセプターからの蛍光が検出されなくなり、代わりにドナーからの蛍光が検出されるようになる。このように、ペプチドの切断により分子から発せられる蛍光の波長が変化するため、この波長変化を利用するとペプチダーゼなどの酵素活性を測定することが可能となる。
これまでに、ＦＲＥＴを利用した酵素活性の測定方法、並びに酵素基質に関する報告は数多く知られている（特許文献１、特許文献２、非特許文献１及び非特許文献２など）。このようなＦＲＥＴを利用した酵素基質及び酵素活性測定方法は、比較的検出感度が高いなどの利点を有する反面、基質の製造及び精製段階が煩雑であり、コストの面においても負担が大きいなど、改善すべき点も多く指摘されている。
Molecules that induce fluorescence resonance energy transfer (FRET) were first synthesized as molecular rulers by Strayer et al., And their effects have been used in various fields since then. The molecule synthesized by Strayer et al. Is a product in which a fluorescent donor and a fluorescent acceptor are introduced at both ends of a peptide having a chain length of 100 mm or less. The acceptor absorbs the fluorescence from the donor and emits fluorescence having a wavelength different from that of the donor. It is something that is emitted. When this peptide is cleaved within the chain, the acceptor cannot absorb the fluorescence from the donor, so that the fluorescence from the acceptor is not detected, and the fluorescence from the donor is detected instead. Thus, since the wavelength of the fluorescence emitted from the molecule is changed by cleaving the peptide, it is possible to measure an enzyme activity such as peptidase using this wavelength change.
So far, many reports on enzyme activity measurement methods and enzyme substrates using FRET have been known (Patent Document 1, Patent Document 2, Non-Patent Document 1, and Non-Patent Document 2, etc.). Such an enzyme substrate and enzyme activity measurement method using FRET have advantages such as relatively high detection sensitivity, but the production and purification steps of the substrate are complicated, and the burden is high in terms of cost. Many points to be improved are also pointed out.
これまでに報告のあるＦＲＥＴ を利用した酵素基質は、比較的低分子のものがほとんどで、ポリマー状の基質で実用的なものはこれまでにほとんど知られていない。例えば、ＦＲＥＴではなく、蛍光寿命の増減を検出して酵素活性を測定するポリマー状の基質の報告は存在するが、感度の点において、ＦＲＥＴには劣ることが予想される（特許文献３）。
以上のようにＦＲＥＴは酵素活性の基質に応用する技術として、極めて優れた技術ではあるが、検出可能なＦＲＥＴを惹起する基質を簡易にしかも低コストで製造する実用的な方法は、これまでの所知られていない。
Enzyme substrates using FRET that have been reported so far are mostly low-molecular-weight substrates, and practically practical polymeric substrates have not been known so far. For example, although there is a report of a polymer-like substrate that detects enzyme activity by detecting increase / decrease in fluorescence lifetime instead of FRET, it is expected to be inferior to FRET in terms of sensitivity (Patent Document 3).
As described above, although FRET is an extremely excellent technique as a technique applied to an enzyme-active substrate, a practical method for producing a substrate that induces a detectable FRET simply and at low cost has been proposed so far. Not known.
従来、ＦＲＥＴを利用した酵素基質は、低分子性のものが多く、その製造のために多くの工程と費用が必要とされていた。そのため、酵素活性の検出感度において優れているＦＲＥＴを、酵素基質として汎用性の高い技術として使用する上で、より簡易でコストも低い製造方法に改善する必要があった。
そこで、本発明は、ＦＲＥＴを利用したポリマー形態の酵素基質、及び、その製造方法の提供を目的とするものである。
Conventionally, many enzyme substrates using FRET have low molecular weight, and many processes and costs have been required for their production. Therefore, when using FRET, which is excellent in enzyme activity detection sensitivity, as a highly versatile technique as an enzyme substrate, it has been necessary to improve the production method to be simpler and less costly.
Accordingly, the object of the present invention is to provide a polymer-form enzyme substrate using FRET and a method for producing the same.
本発明者らは、ＦＲＥＴ効果を利用した酵素基質の開発を行っていたところ、ＦＲＥＴの蛍光ドナー及び蛍光アクセプターを担持したモノマーからなるポリマーの合成に成功し、さらに、該ポリマーが酵素基質となり得ることを見出して本発明を完成させた。
ＦＲＥＴの蛍光ドナーと蛍光アクセプターを担持させたモノマーを使用し、ラジカル重合反応などを利用すると、１つの工程で短時間のうちにＦＲＥＴ感受性ポリマーを調製することが可能である。ここで調製されたポリマーには、酵素の認識部分を介して蛍光ドナー又はアクセプターが結合するモノマーが構成要素として含まれている。該ポリマーを基質として酵素活性を測定すると、非常に良好なＦＲＥＴ効果を確認することができた。
The present inventors have been developing an enzyme substrate using the FRET effect, and succeeded in synthesizing a polymer composed of a monomer carrying a fluorescent donor and a fluorescent acceptor of FRET. Further, the polymer can be an enzyme substrate. As a result, the present invention was completed.
When a monomer carrying a FRET fluorescent donor and a fluorescent acceptor is used and a radical polymerization reaction or the like is used, it is possible to prepare a FRET-sensitive polymer within a short time in one process. The polymer prepared here contains, as a constituent, a monomer to which a fluorescent donor or acceptor is bonded via a recognition moiety of the enzyme. When the enzyme activity was measured using the polymer as a substrate, a very good FRET effect could be confirmed.
すなわち、本発明は、以下の式（Ｉ）のポリマー形態の酵素基質である。
（式（Ｉ）中、Ａ、Ｂ又はＣのいずれか１つにＦＲＥＴの蛍光ドナーが結合しており、他の２つのいずれか１つにＦＲＥＴの蛍光アクセプターが結合しており、該蛍光ドナー又は該蛍光アクセプターのいずれか１つは、酵素の基質部分を介して結合しており、ｘ、ｙ及びｚは１以上の整数である。ただし、蛍光ドナー及び蛍光アクセプターのいずれも結合していないＡ、Ｂ又はＣに対応するｘ、ｙ又はｚは、０もしくは１以上の整数である。）
さらに、本発明は、ＦＲＥＴの蛍光ドナーが結合している重合性化合物、ＦＲＥＴの蛍光アクセプターが結合している重合性化合物、並びに、ＦＲＥＴの蛍光ドナー及びＦＲＥＴの蛍光アクセプターのいずれも結合していない重合性化合物を共重合させてポリマー形態の酵素基質を１工程で、一気に製造する方法であって、蛍光ドナー又は蛍光アクセプターのいずれか１つの結合が酵素基質部分を介する結合である、ポリマー形態の酵素基質の製造方法である。
That is, the present invention is an enzyme substrate in the polymer form of the following formula (I).
(In the formula (I), a fluorescent donor of FRET is bound to any one of A, B or C, and a fluorescent acceptor of FRET is bound to any one of the other two, and the fluorescent donor Alternatively, any one of the fluorescent acceptors is bound through the substrate portion of the enzyme, and x, y, and z are integers of 1 or more, provided that neither the fluorescent donor nor the fluorescent acceptor is bound. X, y, or z corresponding to A, B, or C is 0 or an integer of 1 or more.)
Furthermore, the present invention relates to a polymerizable compound to which a fluorescent donor of FRET is bound, a polymerizable compound to which a fluorescent acceptor of FRET is bound, and neither a fluorescent donor of FRET nor a fluorescent acceptor of FRET is bound. A method for producing a polymer form of an enzyme substrate in a single step by copolymerizing a polymerizable compound, wherein either one of a fluorescent donor or a fluorescent acceptor is bound via an enzyme substrate moiety. A method for producing an enzyme substrate.
本発明の基質は、ＦＲＥＴ効果を利用して酵素活性を検出するものであり、共重合反応により煩雑な工程を経ることなく製造することが可能であり、また、製造コストも安価に抑えることができる。 The substrate of the present invention detects enzyme activity using the FRET effect, and can be produced without a complicated process by a copolymerization reaction, and the production cost can be kept low. it can.
本発明の実施形態の１つは、ＦＲＥＴの蛍光ドナーが酵素基質部分を介し、又は介さずに結合した重合性化合物と、ＦＲＥＴの蛍光アクセプターが酵素基質部分を介し、又は介さずに結合した重合性化合物を共重合させてポリマー形態の酵素基質を製造する方法、あるいは、ＦＲＥＴの蛍光ドナー及びＦＲＥＴの蛍光アクセプターのいずれも結合していない重合性化合物をさらに加えて共重合させたポリマー形態の酵素基質を製造する方法である。
これらの重合性化合物をモノマーとして共重合させる場合、当業者によって容易に選択される重合方法であれば、いかなるものであってもよいが、重合反応を可能な限り短時間で、一気に進行させることができる重合方法が望ましい。重合方法として、例えば、付加重合反応（ラジカル重合反応、イオン重合反応）、開環重合反応、重縮合反応、重付加反応、付加縮合反応などが挙げられ、特に、ラジカル重合反応が簡便で好ましい方法である。また、本発明において使用される重合性化合物は、重合可能な官能基がモノマー内に存在し、溶媒に可溶であれば、いかなる化合物であってもよく、このような化合物は当業者により容易に選択可能なものを、目的に応じて適宜選択することができる。特に限定はしないが、本発明のモノマーとして例を挙げるとすれば、例えば、スチレンなどの芳香族ビニル系モノマー、アクリルアミドなどのアクリル系モノマー、メタクリル系モノマー、アクリロニトリル、酢酸ビニルなどを挙げることができる。モノマーとして脂溶性のもの、例えば、スチレンなどを使用すると脂溶性の高いポリマーへの誘導が可能になるため、このように製造された酵素基質は、プラスチックなどの支持体に対して疎水性相互作用により、予め固定することも可能であり、酵素活性の測定キットなどの作製にも利用できる。
In one embodiment of the present invention, a polymerizable compound in which a fluorescent donor of FRET is bound via or not via an enzyme substrate moiety, and a polymerization in which a fluorescent acceptor of FRET is bound via or not via an enzyme substrate moiety. A method for producing an enzyme substrate in the form of a polymer by copolymerizing a functional compound, or an enzyme in the form of a polymer obtained by further adding a polymerizable compound to which neither a FRET fluorescent donor nor a FRET fluorescent acceptor is bound A method for producing a substrate.
In the case of copolymerizing these polymerizable compounds as monomers, any polymerization method can be used as long as it is easily selected by those skilled in the art, but the polymerization reaction is allowed to proceed at once in as short a time as possible. Polymerization methods that can be used are desirable. Examples of the polymerization method include addition polymerization reaction (radical polymerization reaction, ionic polymerization reaction), ring-opening polymerization reaction, polycondensation reaction, polyaddition reaction, addition condensation reaction and the like. It is. The polymerizable compound used in the present invention may be any compound as long as a polymerizable functional group is present in the monomer and is soluble in a solvent. Those that can be selected can be selected as appropriate according to the purpose. Although not particularly limited, examples of the monomer of the present invention include aromatic vinyl monomers such as styrene, acrylic monomers such as acrylamide, methacrylic monomers, acrylonitrile, and vinyl acetate. . The use of fat-soluble monomers such as styrene makes it possible to induce a highly liposoluble polymer, so that the enzyme substrate thus produced has a hydrophobic interaction with a support such as plastic. Can be fixed in advance, and can also be used to produce enzyme activity measurement kits and the like.
重合条件は、使用する重合性化合物や重合方法によって異なるが、当業者であれば適切な重合条件を選択することが可能である。また、各重合性化合物の混合比率についても、予め予備的な重合実験などを行うことにより、当業者において容易に決定することができる。例えば、アクリルアミドなどのアクリル系モノマーを重合性化合物として使用した場合、蛍光アクセプターが結合したモノマーと蛍光ドナーが結合したモノマーの混合比率は、アクセプター結合モノマー１に対してモル比で、１〜１００、２〜５０、好ましくは、５〜２０、より好ましくは１０程度である。また、アクセプターとドナーのいずれも結合していない重合性化合物をモノマーとして重合系に添加する場合の混合比率は、ドナー結合モノマーを１に対してモル比で、１〜５０、より好ましくは、５〜２０程度である。 The polymerization conditions vary depending on the polymerizable compound used and the polymerization method, but those skilled in the art can select appropriate polymerization conditions. Also, the mixing ratio of each polymerizable compound can be easily determined by those skilled in the art by conducting preliminary polymerization experiments and the like in advance. For example, when an acrylic monomer such as acrylamide is used as the polymerizable compound, the mixing ratio of the monomer to which the fluorescent acceptor is bonded and the monomer to which the fluorescent donor is bonded is 1 to 100 in terms of a molar ratio with respect to the acceptor-binding monomer 1. It is 2-50, Preferably, it is 5-20, More preferably, it is about 10. In addition, the mixing ratio in the case where a polymerizable compound in which neither acceptor nor donor is bonded is added as a monomer to the polymerization system is 1 to 50, more preferably 5 in terms of molar ratio of donor-binding monomer to 1. It is about ~ 20.
本発明のポリマー形態の酵素基質を製造するためのモノマーは、ＦＲＥＴの蛍光ドナー又は蛍光アクセプターが、活性測定対象である酵素による認識部分（つまり、基質部分。例えば、ペプチダーゼであれば切断部位として認識されるアミノ酸配列を含むペプチド部分）を介して結合している重合性化合物である。このようなモノマーを使用すると、ポリマー骨格部分と蛍光ドナー又は蛍光アクセプターのいずれか１つとの間に酵素認識部分が複数介在する構造を持つポリマーが合成される。このようなポリマー形態の酵素基質に対して、例えば、酵素認識部分を切断するような酵素が作用すると、蛍光ドナー又は蛍光アクセプターのいずれかがポリマー骨格部分から分離されることになるため、ＦＲＥＴに変化が生じることになる。このＦＲＥＴ変化を検出することで酵素活性を検出、測定することが可能となる。本発明のポリマー形態の酵素基質は、共重合反応のような容易で一気に反応が進む工程を利用して構築されるものであるため、従来のＦＲＥＴ利用基質に比較して、簡便、かつ、安価に合成することが可能となる。また、本発明の酵素基質を合成するために使用する各モノマーの混合比率を変えることで、ＦＲＥＴの蛍光ドナーと蛍光アクセプターの組成比を容易に変更することができ、酵素活性の測定感度などの調節が可能となる。
ここでＦＲＥＴの蛍光ドナー及び蛍光アクセプターは、ＦＲＥＴを生じさせる蛍光分子ペアであれば如何なる組合せの蛍光分子ペアであってもよく、例えば、ドナー・アクセプターのペアとして、ナフチル基とダンシル基、ＥＤＡＮＳ基とＤＡＢＣＹＬ基などを挙げることができる。より詳細には、例えば、Ａｎａｌｙｔｉｃａｌ Ｂｉｏｃｈｅｍｉｓｔｒｙ ２１８，１−１３（１９９４）などを参照のこと。
The monomer for producing the enzyme substrate of the polymer form of the present invention is a recognition moiety by the enzyme whose activity is to be measured by the fluorescent donor or acceptor of FRET (that is, a substrate moiety. For example, if it is a peptidase, it is recognized as a cleavage site. A polymerizable compound which is bonded via a peptide moiety containing the amino acid sequence to be determined. When such a monomer is used, a polymer having a structure in which a plurality of enzyme recognition portions are interposed between the polymer backbone portion and any one of the fluorescent donor and the fluorescent acceptor is synthesized. For example, when an enzyme that cleaves the enzyme recognition moiety acts on the enzyme substrate in such a polymer form, either the fluorescent donor or the fluorescent acceptor is separated from the polymer backbone part. Change will occur. By detecting this FRET change, the enzyme activity can be detected and measured. Since the enzyme substrate of the polymer form of the present invention is constructed using a process in which the reaction proceeds easily at once, such as a copolymerization reaction, it is simpler and less expensive than the conventional FRET substrate. Can be synthesized. In addition, by changing the mixing ratio of each monomer used for synthesizing the enzyme substrate of the present invention, the composition ratio of the fluorescent donor and the fluorescent acceptor of FRET can be easily changed. Adjustment is possible.
Here, the fluorescent donor and the fluorescent acceptor of FRET may be any fluorescent molecular pair that generates FRET. For example, the donor-acceptor pair includes a naphthyl group, a dansyl group, and an EDANS group. And DABCYL group. For more details, see, for example, Analytical Biochemistry 218, 1-13 (1994).
本発明の製造方法で使用される蛍光ドナーを結合した重合性化合物を、より具体的に示すと、例えば、以下の式（ＩＶ）のようなビニル化合物を挙げることができる。
（式（ＩＶ）中、Ｒ１、Ｒ２及びＲ３が同一又は異なる、水素、塩素、フッ素、メチル基、アセチル基、カルボキシル基、カルボキメチル基、シアノ基であり、Ｒ４は、酵素認識部分を介して、又は介さず結合する蛍光ドナーである）
また、本発明で使用される蛍光アクセプターを結合した重合性化合物を、より具体的に示すと、例えば、以下の式（Ｖ）のようなビニル化合物を挙げることができる。
（式（Ｖ）中、Ｒ５、Ｒ６及びＲ７が同一又は異なる、水素、塩素、フッ素、メチル基、アセチル基、カルボキシル基、カルボキメチル基、シアノ基であり、Ｒ８は、酵素認識部分を介して、又は介さず結合する蛍光ドナーである）
More specifically, the polymerizable compound bound with the fluorescent donor used in the production method of the present invention includes, for example, a vinyl compound represented by the following formula (IV).
(In formula (IV), R 1 , R 2 and R 3 are the same or different, and are hydrogen, chlorine, fluorine, methyl group, acetyl group, carboxyl group, carboxymethyl group, cyano group, and R 4 is enzyme recognition. A fluorescent donor that binds through or without a moiety)
Moreover, when the polymerizable compound to which the fluorescent acceptor used in the present invention is bound is shown more specifically, for example, a vinyl compound represented by the following formula (V) can be mentioned.
(In formula (V), R 5 , R 6 and R 7 are the same or different and are hydrogen, chlorine, fluorine, methyl group, acetyl group, carboxyl group, carboxymethyl group, cyano group, and R 8 is enzyme recognition. A fluorescent donor that binds through or without a moiety)
上記式（ＩＶ）及び式（Ｖ）で示されるような化合物とアクリルアミドのような重合性化合物を使用して重合反応を行った場合、図１に模式的に示されるようなポリマー基質が合成される。図１では、例として、励起波長２９０ｎｍで３４０ｎｍの発光を生じる蛍光ドナーを結合させたモノマー、励起波長３４０ｎｍで５４０ｎｍの発光を生じる蛍光アクセプターを結合させたモノマー及びアクリルアミドを用いて合成した酵素基質を示してある。合成されたポリマーは、励起波長２９０ｎｍで発光させるとドナーからは３４０ｎｍの発光が生じるが、この発光波長は、アクセプターの励起波長と重なるため、ＦＲＥＴ効果により５４０ｎｍの発光がアクセプターから観測される。ここで、蛍光ドナーが任意の加水分解酵素の認識部分を介してポリマー基質に結合している場合、このポリマー基質に加水分解酵素を作用させると酵素認識部分が切断されてドナーが脱離する。その結果、ＦＲＥＴ効果が消失して、代わりにドナー本来の発光（３４０ｎｍ）が観測されるようになる。
以上のように、本発明のポリマー形態の酵素基質を使用すれば、観測される蛍光の発光波長の変化を指標にして酵素活性を検出、測定することができる。
When a polymerization reaction is performed using a compound represented by the above formulas (IV) and (V) and a polymerizable compound such as acrylamide, a polymer substrate schematically shown in FIG. 1 is synthesized. The In FIG. 1, as an example, a monomer combined with a fluorescent donor that generates 340 nm emission at an excitation wavelength of 290 nm, a monomer combined with a fluorescent acceptor that generates 540 nm emission at an excitation wavelength of 340 nm, and an enzyme substrate synthesized using acrylamide are shown. It is shown. When the synthesized polymer emits light at an excitation wavelength of 290 nm, the donor emits light of 340 nm. Since this emission wavelength overlaps with the excitation wavelength of the acceptor, emission of 540 nm is observed from the acceptor due to the FRET effect. Here, when the fluorescent donor is bonded to the polymer substrate via a recognition moiety of any hydrolase, the enzyme recognition moiety is cleaved and the donor is detached when a hydrolase is allowed to act on the polymer substrate. As a result, the FRET effect disappears and instead the original light emission (340 nm) of the donor is observed.
As described above, when the enzyme substrate of the polymer form of the present invention is used, the enzyme activity can be detected and measured using the observed change in the emission wavelength of fluorescence.
本発明の範囲には、上記製造方法によって製造されるポリマー形態の酵素基質も含まれる。
本発明の酵素基質は、例えば、以下の式（Ｉ）のポリマー形態の化合物として示すことができる。
（式（Ｉ）中、Ａ、Ｂ又はＣのいずれか１つにＦＲＥＴの蛍光ドナーが結合しており、他の２つのいずれか１つにＦＲＥＴの蛍光アクセプターが結合しており、該蛍光ドナー又は該蛍光アクセプターのいずれか１つは、酵素の基質部分を介して結合しており、ｘ、ｙ及びｚは１以上の整数である。ただし、蛍光ドナー及び蛍光アクセプターのいずれも結合していないＡ、Ｂ又はＣに対応するｘ、ｙ又はｚは、０もしくは１以上の整数である）
The scope of the present invention includes an enzyme substrate in a polymer form produced by the above production method.
The enzyme substrate of the present invention can be shown, for example, as a compound in the polymer form of the following formula (I).
(In the formula (I), a fluorescent donor of FRET is bound to any one of A, B or C, and a fluorescent acceptor of FRET is bound to any one of the other two, and the fluorescent donor Alternatively, any one of the fluorescent acceptors is bound through the substrate portion of the enzyme, and x, y, and z are integers of 1 or more, provided that neither the fluorescent donor nor the fluorescent acceptor is bound. X, y or z corresponding to A, B or C is 0 or an integer of 1 or more)
本発明の酵素基質は、ＦＲＥＴ効果を利用した活性測定が可能な酵素であれば如何なる酵素も基質とすることが可能である。特に限定はしないが、例えば、加水分解酵素など、例えば、カルボン酸エステル加水分解酵素（カルボキシルエステラーゼ、コリンエステラーゼ、ホスホリパーゼなど）、リン酸エステル加水分解酵素（ホスファターゼ、ヌクレアーゼなど）などのエステル加水分解酵素、糖加水分解酵素（（α−、β−）アミラーゼ、セルラーゼ、グルコアミラーゼ、グルカナーゼ、グリコシダーゼ、シアリダーゼ、リゾチーム、マルターゼ、ガラクトシダーゼ、サッカラーゼ、グルコシダーゼ、マンノシダーゼ、Ｎ−アセチルヘキソサミニダーゼなど）、ペプチド結合加水分解酵素（ペプチダーゼ、プロテアーゼなど）、エーテル・チオエーテル加水分解酵素、ペプチド以外のＣＮ結合加水分解酵素などの基質として使用することができる。 As the enzyme substrate of the present invention, any enzyme can be used as a substrate as long as the activity can be measured using the FRET effect. Although not particularly limited, for example, hydrolase, for example, ester hydrolase such as carboxylic ester hydrolase (carboxyl esterase, cholinesterase, phospholipase, etc.), phosphate hydrolase (phosphatase, nuclease, etc.), Sugar hydrolase ((α-, β-) amylase, cellulase, glucoamylase, glucanase, glycosidase, sialidase, lysozyme, maltase, galactosidase, saccharase, glucosidase, mannosidase, N-acetylhexosaminidase, etc.), peptide bond hydrolysis It can be used as a substrate for degrading enzymes (peptidases, proteases, etc.), ether / thioether hydrolases, and CN bond hydrolases other than peptides.
例えば、アミラーゼの基質として、以下の式（ＶＩ）で示される化合物を例示することができる。式（ＶＩ）のポリマー化合物は、Ａに対応するモノマーとして式（ＩＩ）の化合物を使用し、Ｃに対応するモノマーとして式（ＩＩＩ）の化合物を使用し、Ｂに対応するモノマーとしてアクリルアミドを使用し、本発明の製造方法によって合成することができる。ここで、式（ＩＩ）の化合物には、アミラーゼの基質部分を介して蛍光ドナーが結合しており、式（ＩＩＩ）の化合物には蛍光アクセプターが結合している。
本発明の酵素基質は、適当な容器又はパック中に使用説明書と共に酵素アッセイ用のキットとして提供することができる。本発明のキットとして供給される場合、本発明の酵素基質の他、酵素活性を測定するために必要なバッファーなどの試薬等が含まれていてもよい。酵素基質の安定性を保持するために、当業者によって適宜選択される保存方法によって、本発明の酵素基質をキット中に収納することが望ましい。本発明の酵素基質を収納する容器としては、酵素基質の物理化学的な安定性が保持されるような材質からなるものであればいかなる形状のものであってもよい。例えば、９６穴プレートなどのプラスチック製の基板等に分注し、場合によっては、該基質を基板状に固定（疎水性相互作用などにより）し、必要に応じて乾燥させた状態で提供してもよい。また、キットに使用説明書が添付される場合、その使用説明は、紙又は他の材質上に印刷されて供給されてもよく、フロッピー（登録商標）ディスク、ＣＤ−ＲＯＭ、ＤＶＤ−ＲＯＭ、Ｚｉｐディスク、ビデオテープ、オーディオテープなどの電気的又は電磁的に読み取り可能な媒体として供給されてもよい。あるいは、キットの製造者によって指定され又は電子メール等で通知されるウェブサイトに掲載されていてもよい。 The enzyme substrate of the present invention can be provided as a kit for enzyme assay together with instructions for use in a suitable container or pack. When supplied as a kit of the present invention, in addition to the enzyme substrate of the present invention, reagents such as buffers necessary for measuring enzyme activity may be included. In order to maintain the stability of the enzyme substrate, it is desirable to store the enzyme substrate of the present invention in the kit by a storage method appropriately selected by those skilled in the art. The container for storing the enzyme substrate of the present invention may have any shape as long as it is made of a material capable of maintaining the physicochemical stability of the enzyme substrate. For example, it is dispensed on a plastic substrate such as a 96-well plate, and in some cases, the substrate is fixed to the substrate (by hydrophobic interaction, etc.) and dried as necessary. Also good. In addition, when the instruction manual is attached to the kit, the instruction manual may be printed on paper or other material and supplied, such as a floppy (registered trademark) disk, CD-ROM, DVD-ROM, Zip. It may be supplied as an electrically or electromagnetically readable medium such as a disk, video tape, audio tape or the like. Alternatively, it may be posted on a website designated by the manufacturer of the kit or notified by e-mail or the like.
以下の実施例は、本発明の酵素基質として、アミラーゼ活性測定用基質の合成例とアミラーゼ活性測定結果を示すものである。本発明の実施例は、あくまでも例示にすぎず、本発明の範囲を限定するものではない。 The following examples show synthesis examples of amylase activity measurement substrates and amylase activity measurement results as enzyme substrates of the present invention. The embodiments of the present invention are merely examples, and do not limit the scope of the present invention.
１．アミラーゼ基質合成
１−１. マルトテトラオース誘導体（ドナー）の合成
マルトテトラオース（１）を完全アセチル化した後、ＢＦ３・Ｅｔ２Ｏをプロモータして４−ペンテン−１−オールとのグリコシル化により、ペンテニル基をアノマー位に導入した。Ｚｅｍｐｌeｎ（ツェンプレン）条件下、脱アセチル化し、非還元末端の４位と６位をナフチリデン保護してから、残った水酸基を完全アセチル化した。さらに、Ｍｅ３Ｎ・ＢＨ３とＡｌＣｌ３を用いて選択的還元開裂させ、Ｚｅｍｐｌeｎ条件下で脱アセチル化した。ＵＶ照射下、２−アミノエタンチオールとラジカル付加反応をした後、アクリロイル化、精製のため完全アセチル化、さらに脱アセチル化して重合性蛍光ドナー（２）の合成を達成した（スキーム１）。
１−２．ダンシルクロライド誘導体（アクセプター）の合成
ダンシルクロライド（３）により、エチレンジアミンをダンシル化しアミン４へと誘導後、アクリロイル化して重合性蛍光アクセプター（５）を合成した（スキーム２）。
１−３．ドナーとアクセプターの重合反応
蛍光ドナー（２）、蛍光アクセプター（５）、およびアクリルアミドをＡＰＳ−ＴＥＭＥＤによるラジカル共重合反応によりポリマー（６）へと誘導した（スキーム３）。
合成例のポリマーの組成を表１に示す。
２．アミラーゼ活性の測定
上記のように合成した基質を１００μｌのＤＭＳＯに溶解させた後、１０μｌの溶液を６ｍｌのＨＥＰＥＳバッファー（ｐＨ７．０）に拡散したものを基質溶液とし、活性測定には、そのうち３ｍｌ使用した。また、酵素溶液は、０．１ｍｇのアミラーゼを１ｍｌのＨＥＰＥＳに溶解させたものを１０μｌ使用した。基質溶液と酵素溶液を混合したのち、３７℃にて、経時的に蛍光スペクトルを測定した。その結果、時間の変化と共に５２０ｎｍの蛍光アクセプター由来の蛍光が減少し、３４０ｎｍ付近の蛍光ドナー由来の蛍光が増加することが明らかとなった。また、この蛍光スペクトルの変化は、３７０ｎｍ付近に等発光点が観測されたことから、単なる蛍光消光や希釈による現象ではないことも確かめられた（図２）。
2. Measurement of amylase activity The substrate synthesized as described above was dissolved in 100 μl of DMSO, and then 10 μl of solution was diffused in 6 ml of HEPES buffer (pH 7.0) to form a substrate solution. used. As the enzyme solution, 10 μl of 0.1 mg amylase dissolved in 1 ml of HEPES was used. After mixing the substrate solution and the enzyme solution, the fluorescence spectrum was measured over time at 37 ° C. As a result, it became clear that the fluorescence derived from the fluorescence acceptor at 520 nm decreased with the change in time, and the fluorescence derived from the fluorescent donor near 340 nm increased. In addition, it was confirmed that the change in the fluorescence spectrum was not a phenomenon caused by simple fluorescence quenching or dilution because an equiemission point was observed near 370 nm (FIG. 2).
本発明によれば、ポリマー形態の酵素基質を簡易、かつ、安価に製造することができるため、酵素活性の検出に要するコストを削減することが可能となる。その結果、酵素活性の検出手段を利用する各分野に多大な貢献をすることが期待される。 According to the present invention, since the enzyme substrate in a polymer form can be produced easily and inexpensively, the cost required for detecting the enzyme activity can be reduced. As a result, it is expected to make a great contribution to each field using the enzyme activity detection means.
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