Immobilization of Penicillin G Acylase on Vinyl Sulfone-Agarose: An Unexpected Effect of the Ionic Strength on the Performance of the Immobilization Process

Abstract

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.

Boosting the stability of β-galactosidase immobilized onto soy-protein isolate-glutaraldehyde-functionalized carrageenan beads

“…Hence, it could be concluded that the thermal stability of the glycine-treated iβGL was still impaired. On the other hand, blocking the remaining vinyl-sulfone moieties of the divinyl-sulfone-functionalized agarose via incubation in glycine boosted the thermal stability of immobilized lipase (Dos Santos et al 2015 ) and the stability of immobilized penicillin G acylase (Da Rocha et al 2022 ). Moreover, utilizing glycine to block the immobilizer remaining epoxy moieties boosted the thermal stabilities of the ES-105 immobilized transaminase (Jia et al 2020 ) and the Eupergit C iβGL (Torres and Batista-Viera 2012 ) (Table 1 ).…”

“…The blocking boosted the thermal stability of the immobilized-blocked enzyme preparation (Dos Santos et al 2015 ). Blocking via incubation in glycine or ethanolamine was also reported to boost the stability of the penicillin G acylase immobilized onto vinyl-sulfone agarose (Da Rocha et al 2022 ). Such stability enhancements were probably consequences of inhibiting the uncontrolled enzyme-immobilizer interactions.…”

Enzyme Immobilization

“…The last paper is on the immobilization of penicillin G acylase [ 23 , 24 ] on vinyl sulfone activated supports [ 25 ]. These supports have been recognized recently as very well suited to yield intense multipoint covalent attachment [ 3 ], and the enzyme had previously immobilized/stabilized on glyoxyl and epoxy supports [ 26 , 27 ].…”

“…However, the immobilization failed on vinyl sulfone agarose beads. The authors were able to force the enzyme immobilization using high ionic strength and enabling the hydrophobic enzyme adsorption on the moderately hydrophobic support surface, achieving very good stabilization results after optimization of the multi-point covalent immobilization [ 25 ].…”