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

Morellon-Sterling, RobertoBolívar Bolívar, Juan ManuelFernandez-Lafuente, Roberto2025-01-162025-01-162022-11Roberto Morellon-Sterling, Juan M. Bolivar, Roberto Fernandez-Lafuente, 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, International Journal of Biological Macromolecules, Volume 220, 2022, Pages 1155-1162, ISSN 0141-8130, https://doi.org/10.1016/j.ijbiomac.2022.08.155.0141-813010.1016/j.ijbiomac.2022.08.155https://hdl.handle.net/20.500.14352/114622The 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 β-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 β-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.engAttribution-NonCommercial-ShareAlike 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-sa/4.0/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 supportsworking paperhttps://www.sciencedirect.com/science/article/pii/S014181302201858X?via%3Dihubopen access66.0577.1Reversible Cys protectionCysteinyl enzymes immobilizationEnzyme stabilizationIngeniería químicaBioquímica (Química)2302 Bioquímica3303 Ingeniería y Tecnología Químicas