Stabilization of Multimeric Enzymes Via Immobilization and Further Cross Linking With Aldehyde Dextran

Cesar Mateo, Benevides C. C. Pessela, Manuel Fuentes, Rodrigo Torres, Lorena Betancor, Aurelio Hidalgo, Gloria Fernández-Lorente, Roberto Fernandez-Lafuente, and Jose M. Guisan

Summary

Subunit dissociation of multimeric proteins is one of the most important causes of inac-tivation of proteins having quarternary structure, making these proteins very unstable under diluted conditions. A sequential two-step protocol for the stabilization of this protein is proposed. A multisubunit covalent immobilization may be achieved by performing very long immobilization processes between multimeric enzymes and porous supports composed of large internal surfaces and covered by a very dense layer of reactive groups. Additional cross-linking with polyfunctional macromolecules promotes the complete cross-linking of the subunits to fully prevent enzyme dissociation. Full stabilization of multimeric structures has been physically shown because no subunits were desorbed from derivatives after boiling them in SDS. As a functional improvement, these immobilized preparations no longer depend on the enzyme.

Key Words: Solid-phase chemical modification; aldehyde dextran cross-link; stabilization of quaternary structures; multisubunit immobilization.

1. Introduction

Protein immobilization may be of use not only to the re-use or continuous use of industrial enzymes. Immobilization and subsequent postimmobilization techniques can be useful for greatly increasing the activity-stability properties of industrial enzymes (1,2). In this chapter, the application of these techniques for the development of a general strategy to stabilize the quaternary structure of multimeric enzymes is discussed.

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