Co-immobilization of lipase and laccase on agarose-based supports via layer-by-layer strategy: effect of diffusional limitations.

Co-immobilized enzymes present desirable advantages to performing cascade reactions when employed in one-pot processes, mainly because of the shorter lag times owing to better reagent diffusion across the active sites of the different enzymes. But high product yields require high enzyme loads, usually achieved with high surface area and porous supports, where diffusional limitations may be introduced or increased. Heterogeneous biocatalysts were prepared by co-immobilizing Pseudomonas fluorescens lipase (PFL) and Aspergillus sp. Laccase (Novozymes 51003) on two agarose-based supports, DEAE-Sepharose and Octyl-agarose. A layer-by-layer strategy was used by adding polyethyleneimine (PEI) to allow the attachment of the upper enzyme layer onto the lower adjacent layer. A final step, crosslinking with glutaraldehyde, was performed to prevent enzyme leaching under harsh environmental conditions. The heterogeneous biocatalysts showed lower temperature dependence and a significant increase in thermal stability at 50°C. No internal mass transfer effects were observed when a low protein load was...