4.6 Article

Synergy of Ion Exchange and Covalent Reaction: Immobilization of Penicillin G Acylase on Heterofunctional Amino-Vinyl Sulfone Agarose

Journal

CATALYSTS
Volume 13, Issue 1, Pages -

Publisher

MDPI
DOI: 10.3390/catal13010151

Keywords

ion exchanger; vinyl sulfone-activated support; heterofunctional supports; synergy in enzyme immobilization; covalent enzyme immobilization; penicillin G acylase

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In this study, MANAE-vinyl sulfone (VS) agarose beads were found to be a better support for immobilizing PGA enzyme compared to agarose-VS beads. The modified beads allowed for rapid and covalent immobilization of PGA, resulting in improved stability compared to low ionic strength VS-agarose.
Agarose-vinyl sulfone (VS) beads have proven to be a good support to immobilize several enzymes. However, some enzymes are hardly immobilized on it. This is the case of penicillin G acylase (PGA) from Escherichia coli, which is immobilized very slowly on this support (less than 10% in 24 h). This enzyme is also not significantly adsorbed in aminated MANAE-agarose beads, an anionic exchanger. In this study, MANAE-agarose beads were modified with divinyl sulfone (DVS) to produce MANAE-vinyl sulfone (VS) agarose beads. When PGA was immobilized on this support, the enzyme was fully immobilized in less than 1.5 h. PGA cannot be released from the support by incubation at high ionic strength, suggesting that the enzyme was rapidly immobilized in a covalent fashion. Considering that the amount of reactive VS groups was only marginally increased, the results indicated some cooperative effect between the anion exchange on the amine groups of the support, probably as the first step of the process, and the covalent attachment of the previously adsorbed PGA molecules. The covalent reaction of the previously adsorbed enzyme molecules proceeds much more efficiently than that of the free enzyme, due to the proximity of the reactive groups of the support and the enzyme. Finally, the steps of immobilization, incubation, and blocking with different agents were studied to determine the effects on final activity/stability. The stability of PGA immobilized on this new catalyst was improved with respect to the VS-agarose prepared at low ionic strength.

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