Switch off/switch on of a cysteinyl protease as a way to preserve the active catalytic group by modification with a reversible covalent thiol modifier: Immobilization of ficin on vinyl-sulfone activated supports

Autores: Morellon-Sterling, R; Bolivar, JM; Fernandez-Lafuente, R

Artículo.
Int. J. Biol. Macromol.. vol: 220. page: 0141-8130.
Fecha: nov-01. 2022.
Doi: 10.1016/j.ijbiomac.2022.08.155.

Resumen:
The immobilization of ficin (a cysteinyl proteases) on vinyl sulfone agarose produced its almost full inactivation. It was observed that the incubation of the free and immobilized enzyme in beta-mercaptoethanol produced a 20 % of enzyme activity recovery, suggesting that the inactivation due to the immobilization could be a consequence of the modification of the catalytic Cys. To prevent the enzyme inactivation during the immobilization, switching off of ficin via Cys reaction with dipyridyl-disulfide was implemented, giving a reversible disulfide bond that produced a fully inactive enzyme. The switch on of ficin activity was implemented by incubation in 1 M beta-mercaptoethanol. Using this strategy to immobilize the enzyme on vinyl sulfone agarose beads, the expressed activity of the immobilized ficin could be boosted up to 80 %. The immobilized enzyme presented a thermal stabilization similar to that obtained using ficin-glyoxyl-agarose beads. This procedure may be extended to many enzymes containing critical Cys, to permit their immobilization or chemical modification..