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

Abstract

Multifunctional supports containing epoxy groups are here proposed as a second generation of activated supports for covalent immobilization of enzymes following the epoxy chemistry on any type of support (hydrophobic or hydrophilic ones) under very mild experimental conditions (e.g., low ionic strength, neutral pH values, and low temperatures). These multifunctional supports have been easily prepared by modifying a small fraction (10-20%) of the epoxy groups contained in commercial epoxy supports. In this way, additional groups that were able to physically adsorb proteins (e.g., cationic or anionic 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 structural features). Then, "intramolecular" covalent linkages between some nucleophilic groups of the adsorbed enzyme (e.g., amino, thiol, or hydroxy groups) and the dense layer of nearby epoxy groups on the support are established. This two-step covalent immobilization dramatically improves the very low reactivity of epoxy groups toward nonadsorbed proteins. In this way, all other relevant practical advantages of epoxy groups for protein immobilization (their high stability and their ability to form very strong linkages with several nucleophilic enzyme residues with minimal chemical modification) can be an object of universal exploitation. The use of these new multifunctional supports exhibits important advantages regarding immobilization of enzymes previously adsorbed on hydrophobic homofunctional epoxy supports: (i) hydrophilic supports can also be used for immobilization of industrial enzymes; (ii) immobilization can also be carried out at low ionic strength; (iii) every protein contained in crude extracts from Escherichia coli and Acetobacter turbidans can be immobilized by sequentially using a set of different supports; (iv) in most cases, each enzyme has been immobilized on different supports, orientated through different structural features and very likely involving different areas of its surface. For example, three industrial enzymes (penicillin G acylase, lipase, and beta-galactosidase) could be immobilized through different strategies yielding immobilized derivatives with very different activities. The best derivatives preserved 75-100% of activity corresponding to the soluble enzymes used for immobilization, while in some cases a particular immobilization protocol promoted the full inactivation of the enzyme.

Immobilization of enzymes on heterofunctional epoxy supports

“…Table 2 shows some of the most relevant results obtained in our laboratory. Some enzymes are not significantly immobilized on certain supports 19 ; for example, PGA from Escherichia coli is not immobilized on EDA-epoxy supports (this enzyme did not become adsorbed on mild anionic exchangers) 47,48 . The lack of external Cys on the enzyme means that the immobilization on the disulfide-epoxide support was very poor.…”

“…By preparing different heterofunctional epoxy supports [19][20][21] , diverse two-step immobilization protocols can be developed. The overall steps of this procedure are (i) Rapid physical or chemical fixation of protein on the support surface (through different regions of the protein surface) and (ii) Promotion of intramolecular multipoint attachment between the epoxy groups of the support and the nucleophiles placed in the vicinity of the region of the protein participating in the first fixation.…”

“…In fact, this kind of two-step immobilization using hydrophobic supports has already been reported for many enzymes 11,12 . However, protocols for covalent immobilization of enzymes through epoxy groups at low ionic strength or on hydrophilic supports had not yet been reported until the proposal of heterofunctional epoxy supports 19 . Although long-term incubation of the already immobilized proteins under neutral pH may promote the formation of a few covalent linkages between the protein and the epoxy groups in the support 11,12 , the highest intensity of multipoint covalent attachment is achieved via a long-term incubation under alkaline conditions because of the increase in the reactivity of nucleophiles (mainly Lys residues usually present on the protein surface) 13,14,22 (Fig.…”

“…These supports permit immobilization under a very broad range of conditions; the only requirement is that the enzyme must become adsorbed on supports activated with the new groups 19 . Thus, immobilization may be performed at low ionic strength from pH 4 to 10 by combining carboxy-epoxy or amino-epoxy supports.…”

“…Thus, immobilization may be performed at low ionic strength from pH 4 to 10 by combining carboxy-epoxy or amino-epoxy supports. Boronate-epoxy 19 or disulfide-epoxy 20 have an even broader range of conditions under which they may be used.…”