Reactivation strategies by unfolding/refolding of chymotrypsin derivatives after inactivation by organic solvents.
Immobilized enzyme derivatives, in organic media at neutral pH and moderate temperatures, should be mainly and perhaps uniquely inactivated by promotion of conformational changes on their 3D structure. Subsequent irreversible inactivation mechanisms (intermolecular aggregations, chemical modifications, thiol-disulfide exchanges) are thus impossible. However, simple reincubation in aqueous medium of enzymes previously inactivated by solvents usually yields significant but slow and incomplete reactivations. Disruption of incorrect protein structures by denaturing agents (urea, guanidine) is proposed as a new strategy to get rapid, complete and technologically feasible reactivations. By using multipoint immobilized chymotrypsin derivatives, we have evaluated the possibility of unfolding and further refolding of native (non-inactivated) derivatives by different denaturing conditions. After unfolding in 8 M guanidine, derivatives were quickly and completely refolded up to 100% of catalytic activity in 10 minutes. Besides, successive cycles of unfolding and refolding could be exactly reproduced. Finally we checked the possibility to reactivate chymotrypsin derivatives inactivated by dioxane. Simple reincubations in aqueous media yielded a poor reactivation even after 24 hours. However, unfolding in 8 M guanidine enabled complete reactivation in less than 2 hours. From this point of view, by working under 'chemically inert conditions' (moderate pH and temperatures), fully dispersed covalently immobilized enzyme derivatives seem to behave as almost everlasting catalysts despite the very deleterious effect of organic media.
Soler
G
G
Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus Universidad Autónoma, Madrid, Spain.
Bastida
A
A
Blanco
R M
RM
Fernández-Lafuente
R
R
Guisán
J M
JM
D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D004793:Enzyme Reactivators / Q000494:pharmacology*; D004800:Enzymes, Immobilized; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012997:Solvents / Q000494:pharmacology*; D013997:Time Factors
D004793:Enzyme Reactivators; D004800:Enzymes, Immobilized; D012997:Solvents; D002918:Chymotrypsin