Reactivation of penicillin G acylase immobilized in glyoxyl-agarose after inactivation was studied with the purpose of increasing the lifespan of the biocatalyst by simple and reproducible strategies considering unfolding-refolding and direct incubation in reactivation media Reactivation yields were increased with respect to the control (fully aqueous medium) when cosolvents were added to the reactivation medium at concentrations below 50% (v/v) Best results were obtained with 30% (v/v) ethyleneglycol (EG) in both reactivation strategies An increase in reactivation yield from 360 to 62 8% was obtained using the unfolding-refolding strategy while an increase from 500 to 684% was obtained by direct incubation in aqueous media with respect to control Catalytic modulators were also included in the reactivation medium competitive inhibitors (phenylacenc acid and 2-thienylacenc acid) caused a reduction while non-competitive (7-ADCA and 6-APA) caused an increase in reactivation yield Combining cosolvent and catalytic modulators best results in both strategies were obtained with 30% (v/v) EG plus 100 mM 7-ADCA where an increase in reactivation yield from 360 to 960% and from 500 to 980% was achieved with unfolding-refolding and direct incubation in reactivation media respectively Apparent reactivation rate was higher in the case of direct incubation in reactivation media best results being obtained when using 100 mM 7-ADCA and 30% (v/v) EG with an increase with respect to the control (fully aqueous medium with no modulator) from 0 309 h(-1) to 1 129 h(-1) while for unfolding-refolding strategy increase was only from 0 124h(-1) to 0 384 h(-1) Results indicate that direct incubation is a better strategy for penicillin G acylase reactivation and opens up the possibility of significantly increasing the operational lifespan of the biocatalyst by operating the reactor with repeated cycles of reaction and reactivation (c) 2010 Elsevier B V All rights reserved