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
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 imm...
| Autores: | , , |
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| Tipo de documento: | artigo |
| Data de publicação: | 2022 |
| País: | España |
| Recursos: | Universidad Complutense de Madrid (UCM) |
| Repositório: | Docta Complutense |
| Idioma: | inglês |
| OAI Identifier: | oai:docta.ucm.es:20.500.14352/114622 |
| Acesso em linha: | https://hdl.handle.net/20.500.14352/114622 |
| Access Level: | Acceso aberto |
| Palavra-chave: | 66.0 577.1 Reversible Cys protection Cysteinyl enzymes immobilization Enzyme stabilization Ingeniería química Bioquímica (Química) 2302 Bioquímica 3303 Ingeniería y Tecnología Químicas |
| Resumo: | 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. |
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