Abstract
The coimmobilization of six different lipases on octyl agarose beads using an enzyme-multilayer strategy and employing polyethylenimine (PEI) and glutaraldehyde (GLU) as enzyme layer adhesives is shown. Our methodology enables the reuse of the most stable enzymes after the least stable enzyme inactivation takes place. The strategy consisted in immobilizing the most stable enzymes in an irreversible way while leaving the least stable enzymes just physically immobilized. To get the enzyme irreversible immobilization, a GLU treatment of the enzyme adsorbed on PEI was not enough. It must be coated with a new layer of PEI and treated again with GLU. However, this makes the enzyme immobilized in the enzyme-PEI-GLU layer unavailable for release under conditions compatible with the stability of the stable enzymes. This was solved by treating the upper layer of enzyme-PEI-GLU with dextran sulfate, enabling the immobilization and further release of the least stable enzyme, which was from Rhizomucor miehei (RML). Moreover, the GLU treatment had a very dissimilar effect on the enzyme stabilities, converting Lecitase in a stable enzyme while reducing the stability of the enzyme from Thermomyces lanuginosus (TLL). However, a similar treatment of TLL immobilized on octyl agarose had a stabilizing effect. That way, using a proper immobilization strategy and an adequate enzyme ordering, a 5-combi-biocatalyst of coimmobilized stable enzymes could be prepared, leaving RML only physically adsorbed, and the 5-combi-lipase could be reutilized for five series of RML inactivation, desorption, and immobilization of a fresh batch, maintaining more than 90% of the initial activity.