Reuse of anion exchangers as supports for enzyme immobilization: Reinforcement of the enzyme-support multiinteraction after enzyme inactivation

Abstract

β-Galactosidase from Aspergillus oryze has been immobilized on agarose beads coated with polyethyleneimine. The fresh enzyme was released from the support using 500mM NaCl at pH 7. After thermal inactivation or inactivation in the presence of organic solvents, the active enzyme still could be easily released from the support using similar conditions. However, SDS-PAGE analysis of the enzyme contained in the support after enzyme desorption showed that enzyme molecules remained in the support (inactivated enzyme molecules). This effect was stronger on enzyme preparations inactivated in an organic medium. Now the conditions should be greatly strengthen to permit the full enzyme desorption: only after incubation in 2M sodium phosphate at pH 2 and 50°C full release of the enzyme molecules was achieved. This could be repeated several cycles with any difference neither in the immobilization performance nor on the SDS-PAGE analysis. Therefore, the reversibility of the immobilization is a real fact, but recovery of a support fully free of protein molecules is not an easy objective after enzyme inactivation, because the inactivated enzymes seemed to unfold increasing in a great way the interaction with the support, driving to a very strong enzyme-support multi-interaction that difficulty its desorption.