Protocol abstract
Nature Protocols 2, - 1022 - 1033 (2007)
Published online: 26 April 2007 | doi:10.1038/nprot.2007.133
Subject Categories: Chemical modification | Biochemistry and protein analysis
Immobilization of enzymes on heterofunctional epoxy supports
Cesar Mateo1, Valeria Grazu1, Jose M Palomo1, Fernando Lopez-Gallego1, Roberto Fernandez-Lafuente1 & Jose M Guisan1
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.
- Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus UAM, Cantoblanco, Madrid 28049, Spain.
Correspondence to: Roberto Fernandez-Lafuente1 e-mail: rfl@icp.csic.es
Correspondence to: Jose M Guisan1 e-mail: jmguisan@icp.csic.es
nature-products
A-Z product listing
- 2,2'-dipyridyl disulfide(2-PDS)(Sigma Chemical Co.)
- Dithiothreitol (DTT)(Sigma Chemical Co.)
- EDA(Sigma Chemical Company)
- Epoxy Sepabeads(Resindion SRL, Mitsubishi Chemical Corporation)
- Ethylene diamine tetraacetic acid(Sigma Chemical Co.)
- Eupergit C or 250(Degussa, Röhm GmbH & Co. KG)
