TY - JOUR
T1 - Reactivation of immobilized penicillin G acylase
T2 - Influence of cosolvents and catalytic modulators
AU - Miranda, Valeria
AU - Wilson, Lorena
AU - Cárdenas, Constanza
AU - Illanes, Andrés
N1 - Funding Information:
This work was funded by Fondecyt Project 1100323 and 1070361, CONICYT, Chile. Support of CONICYT for a regional graduate scholarship to Ms. Valeria Miranda is also acknowledged.
PY - 2011/1
Y1 - 2011/1
N2 - 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 36.0 to 62.8% was obtained using the unfolding-refolding strategy, while an increase from 50.0 to 68.4% was obtained by direct incubation in aqueous media with respect to control. Catalytic modulators were also included in the reactivation medium: competitive inhibitors (phenylacetic acid and 2-thienylacetic 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 36.0 to 96.0% and from 50.0 to 98.0% 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 0.124 h-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.
AB - 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 36.0 to 62.8% was obtained using the unfolding-refolding strategy, while an increase from 50.0 to 68.4% was obtained by direct incubation in aqueous media with respect to control. Catalytic modulators were also included in the reactivation medium: competitive inhibitors (phenylacetic acid and 2-thienylacetic 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 36.0 to 96.0% and from 50.0 to 98.0% 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 0.124 h-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.
KW - Enzyme reactivation
KW - Immobilized enzyme
KW - Penicillin G acylase
KW - Reaction media engineering
KW - Unfolding-refolding
UR - http://www.scopus.com/inward/record.url?scp=78649332441&partnerID=8YFLogxK
U2 - 10.1016/j.molcatb.2010.09.015
DO - 10.1016/j.molcatb.2010.09.015
M3 - Article
AN - SCOPUS:78649332441
SN - 1381-1177
VL - 68
SP - 77
EP - 82
JO - Journal of Molecular Catalysis B: Enzymatic
JF - Journal of Molecular Catalysis B: Enzymatic
IS - 1
ER -