Penicillin G acylase (PGA) from Escherichia coli was immobilized on vinyl sulfone (VS) agarose. The immobilization of the enzyme failed at all pH values using 50 mM of buffer, while the progressive increase of ionic strength permitted its rapid immobilization under all studied pH values. This suggests that the moderate hydrophobicity of VS groups is enough to transform the VS-agarose in a heterofunctional support, that is, a support bearing hydrophobic features (able to adsorb the proteins) and chemical reactivity (able to give covalent bonds). Once PGA was immobilized on this support, the PGA immobilization on VS-agarose was optimized with the purpose of obtaining a stable and active biocatalyst, optimizing the immobilization, incubation and blocking steps characteristics of this immobilization protocol. Optimal conditions were immobilization in 1 M of sodium sulfate at pH 7.0, incubation at pH 10.0 for 3 h in the presence of glycerol and phenyl acetic acid, and final blocking with glycine or ethanolamine. This produced biocatalysts with stabilities similar to that of the glyoxyl-PGA (the most stable biocatalyst of this enzyme described in literature), although presenting just over 55% of the initially offered enzyme activity versus the 80% that is recovered using the glyoxyl-PGA. This heterofuncionality of agarose VS beads opens new possibilities for enzyme immobilization on this support.

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.