Glyoxyl-ficin: an example where a more intense multipoint covalent attachment may decrease enzyme stability

Abstract

The immobilization of ficin extract (sulfidryl proteases) on glyoxyl agarose beads stabilized it, but there is a clear stability maximum after 3 h of immobilization, being the 24 h biocatalyst less stable when thermally inactivated in presence of air. The incubation of the thermally inactivated biocatalysts in the presence of dithiothreitol permitted to recover almost total activity of the biocatalysts prepared for 24 h of immobilization, while the enzyme immobilized for 3 h recovered less activity. This suggested that the main cause of the faster enzyme inactivation of the 24 h biocatalyst was the Cys oxidation. This was confirmed by inactivating the Cys blocked biocatalysts by using 2,2′-dipyridyldisulfide. Again, a higher percentage of the initial activity (when unblocked by incubation in mercaptoethanol the thermally inactivated biocatalysts) was observed for the 24 h biocatalyst. The use of helium atmosphere enabled to greatly improve the enzyme stability of all immobilized biocatalysts, confirming the highest stability of the biocatalyst immobilized for 24 h. Results suggested that this biocatalyst was in fact the most rigid one, but that the distortion induced by the immobilization produced a higher exposition of the catalytic Cys, and this way, its oxidation becomes faster in the presence of oxygen.