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酶在异功能环氧载体上的固定化。

Immobilization of enzymes on heterofunctional epoxy supports.

作者信息

Mateo Cesar, Grazu Valeria, Palomo Jose M, Lopez-Gallego Fernando, Fernandez-Lafuente Roberto, Guisan Jose M

机构信息

Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus UAM, Cantoblanco, Madrid 28049, Spain.

出版信息

Nat Protoc. 2007;2(5):1022-33. doi: 10.1038/nprot.2007.133.

Abstract

Immobilization of enzymes and proteins on activated supports permits the simplification of the reactor design and may be used to improve some enzyme properties. In this sense, supports containing epoxy groups seem to be useful to generate very intense multipoint covalent attachment with different nucleophiles placed on the surface of enzyme molecules (e.g., amino, thiol, hydroxyl groups). However, the intermolecular reaction between epoxy groups and soluble enzymes is extremely slow. To solve this problem, we have designed "tailor-made" heterofunctional epoxy supports. Using these, immobilization of enzymes is performed via a two-step process: (i) an initial physical or chemical intermolecular interaction of the enzyme surface with the new functional groups introduced on the support surface and (ii) a subsequent intense intramolecular multipoint covalent reaction between the nucleophiles of the already immobilized enzyme and the epoxy groups of the supports. The first immobilization may involve different enzyme regions, which will be further rigidified by multipoint covalent attachment. The design of some heterofunctional epoxy supports and the performance of the immobilization protocols are described here. The whole protocol to have an immobilized and stabilized enzyme could take from 3 days to 1 week.

摘要

将酶和蛋白质固定在活化载体上可以简化反应器设计,并可用于改善某些酶的特性。从这个意义上说,含有环氧基团的载体似乎有助于与置于酶分子表面的不同亲核试剂(如氨基、巯基、羟基)形成非常强烈的多点共价连接。然而,环氧基团与可溶性酶之间的分子间反应极其缓慢。为了解决这个问题,我们设计了“定制”的异功能环氧载体。使用这些载体,酶的固定通过两步过程进行:(i)酶表面与引入载体表面的新官能团之间的初始物理或化学分子间相互作用,以及(ii)随后已固定化的酶的亲核试剂与载体的环氧基团之间强烈的分子内多点共价反应。第一次固定可能涉及不同的酶区域,这些区域将通过多点共价连接进一步刚性化。本文描述了一些异功能环氧载体的设计和固定方案的实施。获得固定化和稳定化酶的整个方案可能需要3天到1周的时间。

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