Multifunctional epoxy supports: A new tool to improve the covalent immobilization of proteins. The promotion of physical adsorptions of proteins on the supports before their covalent linkage

Multifunctional supports containing epoxy groups are here proposed as a sec ond generation of activated supports for covalent immobilization of enzymes following the epoxy chemistry on any type of support (hydrophobic or hydro philic ones) under very mild experimental conditions (e.g., low ionic stren gth, neutral pH values, and low temperatures). These multifunctional suppor ts have been easily prepared by modifying a small fraction (10-20%) of the epoxy groups contained in commercial epoxy supports. In this way, additiona l groups that were able to physically adsorb proteins (e.g., cationic or an ionic groups, metal chelate, phenyl boronate) are generated on the support surface. The covalent immobilization of proteins on these supports proceeds via their initial physical adsorption on the supports (via different struc tural features). Then, "intramolecular" covalent linkages between some nucl eophilic groups of the adsorbed enzyme (e.g., amino, thiol, or hydroxy grou ps) and the dense layer of nearby epoxy groups on the support are establish ed. This two-step covalent immobilization dramatically improves the very lo w reactivity of epoxy groups toward nonadsorbed proteins. In this way, all other relevant practical advantages of epoxy groups for protein immobilizat ion (their high stability and their ability to form very strong linkages wi th several nucleophilic enzyme residues with minimal chemical modification) can be an object of universal exploitation. The use of these new multifunc tional supports exhibits important advantages regarding immobilization of e nzymes previously adsorbed on hydrophobic homofunctional epoxy supports: (i ) hydrophilic supports can also be used for immobilization of industrial en zymes; (ii) immobilization can also be carried out at low ionic strength; ( iii) every protein contained in crude extracts from Escherichia coli and Ac etobacter turbidans can be immobilized by sequentially using a set of diffe rent supports; (iv) in most cases, each enzyme has been immobilized on diff erent supports, orientated through different structural features and very l ikely involving different areas of its surface. For example, three industri al enzymes (penicillin G acylase, lipase, and P-galactosidase) could be imm obilized through different strategies yielding immobilized derivatives with very different activities. The best derivatives preserved 75-100% of activ ity corresponding to the soluble enzymes used for immobilization, while in some cases a particular immobilization protocol promoted the full inactivat ion of the enzyme.