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RevistaJournal of Molecular Catalysis B: Enzymatic,.
Volumen 74 (3-4)

Reactivation of penicillin acylase biocatalysts: Effect of the intensity of enzyme-support attachment and enzyme load

Grupos de investigación:Ingeniería Enzimática
: The influence of the intensity of enzyme-support attachment and enzyme load on the reactivation process has been studied considering three immobilized penicillin G acylase (PGA) biocatalysts: one immobilized by covalent attachment to cyanogen bromide Sepharose (CNBrSep-PGA) and two immobilized by multipoint covalent attachment to glyoxyl agarose with different intensity of attachment (Gx 1-PGA and Gx 2-PGA). The biocatalysts were inactivated in 70% (v/v) dioxane at 5°C down to 25% residual activity. Biocatalysts were then recovered by filtration to remove the inactivation medium and re-incubated in aqueous medium to promote reactivation. The highest level of reactivation (73% recovery of activity with respect to the initial activity before inactivation) was obtained with Gx 1-PGA, corresponding to the biocatalyst with stronger multipoint interactions. For Gx 2-PGA and CNBrSep-PGA, only 33% and 31% of activity was recovered respectively. When the distortion caused by the organic solvent produces a inactive enzyme structure that cannot be significantly recovered by re-incubation in aqueous medium, complete unfolding of the enzyme molecule by chaotropic agents prior to its refolding by incubation in aqueous medium can be used to recover enzyme activity. When this strategy was used, 70% activity was recovered with Gx 1-PGA and Gx 2-PGA, but weakly linked CNBrSep-PGA was completely inactivated after unfolding and no activity was recovered by re-incubation in aqueous medium. With respect to enzyme load, full recovery of enzyme activity was obtained with Gx 1-PGA by reactivation in aqueous medium up to 74 IU/g support. However, at higher enzyme loads recovery was significantly impaired because of intense protein-protein interaction during biocatalyst inactivation and reactivation. 
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