Abstract
It has been found that the enzymes penicillin G acylase from
Escherichia coli (PGA) and lipase from
Bacillus thermocatenulatus (BTL) did not significantly adsorb on highly activated amino-agarose beads at pH 7 (a support where 85–90% of a crude extract of proteins become adsorbed). Moreover, it has been found that these enzymes do not covalently immobilize on highly activated epoxy-agarose beads at pH 7. However, both enzymes slowly immobilize on heterofunctional supports having a high density of amino–epoxy groups. The immobilized enzymes retain a high percentage of activity (more than 90% for PGA and 60% for BTL). On the other hand, the immobilization of a crude extract of proteins on amino–epoxy supports under conditions where only a limited protein ionic exchange was permitted (by using high ionic strength or lowly activated supports), also permitted a similar high immobilization yield of the proteins. Similarly, glutamate dehydrogenase (GDH) and β-galactosidase from
Thermus thermophilus can be fully immobilized under conditions where less than 20% of these enzymes can be ionically exchanged in the aminated support. The results suggested that the percentage of proteins that may be physically adsorbed on the support becomes irreversibly immobilized by the covalent reaction between the nucleophilic groups in the protein surface and the very near epoxy groups of the support (in an almost intramolecular reaction). Thus, using these supports, it is possible to immobilize almost all the proteins by anionic exchange, that is, the area with the highest density in anionic groups. In many cases, this region could not correspond to the protein regions usually utilized to immobilize proteins. This way, it is possible to achieve, in a very simple fashion and without modifying the protein, new orientations of some immobilized enzymes and proteins.