Electronic Journal of Biotechnology ISSN: 0717-3458
© 2000 by Universidad Católica de Valparaíso -- Chile
ORAL PRESENTATION

Stabilization of Immobilized Enzymes Against Organic Solvents: Generation of "Polymeric Salts" Surrounding Enzyme Molecules by Combined use of Cationic and Anionic Random Coil Polymers

J.M. Guisán*
Royal Institute of Technology, Department of Biotechnology, (KTH), Sweden
E-mail: martinl@biochem.kth.se

R. Fernández-Lafuente
Royal Institute of Technology, Department of Biotechnology, (KTH), Sweden
E-mail: martinl@biochem.kth.se

G. Fernández-Lorente
Royal Institute of Technology, Department of Biotechnology, (KTH), Sweden
E-mail: martinl@biochem.kth.se

C. Mateo
Royal Institute of Technology, Department of Biotechnology, (KTH), Sweden
E-mail: martinl@biochem.kth.se

O. Abian
Royal Institute of Technology, Department of Biotechnology, (KTH), Sweden
E-mail: martinl@biochem.kth.se

*Corresponding Author

Keywords: enzyme stabilization by polymeric salts. enzymes in organic media, penicillin G acylase


Oral Presentation

The combination of cationic (polyethyleneimine) and anionic (dextran-sulfate, carragenate, etc) random coil polymers is here proposed to promote dramatic alterations in the nano-environment of immobilized enzymes without (or with minimal) modification of the enzyme structure. Two different approaches will be discussed:

a.- enzymes are co-immobilized with a high amount of polyethyleneimine on the same support surface. Polyethyleneimine polymers much larger than the enzyme molecules were used in order to completely cover the immobilized enzyme. Then, dextran-sulfate is added to the immobilized derivative in order to form a ionic composite with immobilized polyethyleneimine. The immobilized enzyme becomes buried inside this nano-environment of "polymeric salts" and thus protected from the deleterious effect of organic solvent. Enzyme immobilization can be performed by multipoint covalent attachment and hence stabilization of immobilized enzyme molecules can be promoted by the additive effect of "3D enzyme rigidification" plus "hydrophilization of enzyme nano-environment".

b.- the soluble enzymes are "entrapped" inside beds of polycationic-polyanionic composites. Delivering of very small drops of a solution of carragenate on a concentrated solution of polyethyleneimine promotes the formation of very stable "polymeric salt" beads. The presence of soluble enzyme in the carragenate solution allows its "entrapment" inside these highly hydrophilic beads.

By using these approaches, immobilized derivatives of penicillin G acylase from E.coli (PGA) and of Pig Liver esterase were prepared. In both cases, immobilized derivatives were very stable in the presence of organic solvents (inactivation constants were diminished by several orders of magnitude). For example, native PGA or non-stabilized derivatives were rapidly inactivated even in the presence of 40 % of organic cosolvent. On the contrary, these new stabilized derivatives are now very stable in the presence of 95 % of organic cosolvents as well as in the presence of anhydrous organic media. Because of this high stability, new interesting PGA biotransformations (e.g. thermodynamically controlled antibiotic synthesis in organic media) can be now performed.

Supported by UNESCO / MIRCEN network
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