Optimizing the activation of agarose beads with divinyl sulfone for enzyme immobilization and stabilization

The focus of the present work is to find the optimal conditions for the activation of agarose beads with divinyl sulfone (DVS). The reactivity of the vinyl sulfone groups in the support was checked by the support capacity to react with ethylamine; via elemental analysis. In addition, trypsin was used as a model enzyme to test the immobilization and stabilization capabilities of the different supports. The higher the pH, the more vinyl sulfone groups are incorporated into the support, but lower reactivity versus ethylamine is observed. Too long activation times led to similar results. A N/S ratio of 1 means that all vinyl sulfone groups were reactive, and it was always lower than tis figure. The N in the support was 50 % of the amount observed for glyoxyl supports activated with ethylenediamine, suggesting the VS polymerization may be a likely explanation for this result. The higher N/S ratio in the support (modified with ethylamine), the higher the obtained stabilization, very likely by the lower polymerization of the vinyl sulfone on the support. We propose 360 mM divinyl sulfone, at pH 11.5 and 2 h as optimal conditions to reach the highest enzyme stabilization by immobilization in this support.