RT Generic
T1 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
A1 Morellon-Sterling, Roberto
A1 Bolívar Bolívar, Juan Manuel
A1 Fernandez-Lafuente, Roberto
AB 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 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.
PB Elsevier
SN 0141-8130
YR 2022
FD 2022-11
LK https://hdl.handle.net/20.500.14352/114622
UL https://hdl.handle.net/20.500.14352/114622
LA eng
NO Roberto 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.
NO Agencia Estatal de Investigacion, Ministerio de Ciencia e Innovaci´on from Spanish Government (PID2021-122398OB-I00)
NO FPU fellowship (Ministerio de Educacion
DS Docta Complutense
RD 6 abr 2025