Commercial epoxy supports are perhaps the most suitable ones for immobilization of industrial enzymes: i.- epoxy groups are very stable during storage and transportation from producers to consumers, ii.- they are able to react with several groups of the proteins directly yielding very stable links (secondary amine, tioether, ether bonds), iii.- immobilization protocols are quite simple, etc. The immobilization of enzymes on these supports requires the previous physical adsorption (usually hydrophobic) of the enzyme. Therefore, commercial epoxy supports usually have a fairly hydrophobic structure. The presence of hydrophobic surfaces very close to the immobilized enzyme molecules usually promotes important destabilizing effects. For this reason, randomly immobilized enzyme-epoxy derivatives may be even much less stable than native enzymes.

In this communication, a new strategy to greatly increase the stability of enzymes immobilized on hydrophobic epoxy supports is discussed. A three step immobilization protocol is proposed: a.- a first mild immobilization of the enzyme at neutral pH in the presence of high concentrations of phosphate buffer, b.- a subsequent promotion of a very intense multipoint covalent attachment between immobilized enzyme molecules and highly activated epoxy-supports by evaluating the long-term incubation of enzyme-epoxy derivatives under different experimental conditions (pH value, ionic strength, presence of additives) and c.- a final blocking the remaining epoxy supports with highly hydrophilic compounds (e.g. ionic amino acids like lysine, glutamate, glycine etc ) in order to achieve a dramatic diminution of the hydrophobicity of the support surface.

Penicillin G acylase from E.coli was used to exemplify the advantages of this combined stabilizing approach. The best PGA derivatives were more than 1.000 fold more stable than soluble enzyme and they preserving 70% of the initial activity. On the contrary some simply immobilized PGA-epoxy derivatives were even less stable than soluble enzyme.