Abstract
Enzyme immobilisation is of great importance for the fabrication of heterogeneous biocatalysts, as it allows the stabilisation of proteins using a solid support. Moreover, it permits their reuse in continuous and discontinuous reactors. The behaviour of enzymes at the interface with the materials where they are supported is not well understood during operational conditions. Here, we use X-ray fluorescence (XRF) imaging to study the changes in the overall structure of a heterogeneous biocatalyst formed by two unmodified metalloenzymes (a copper-dependent laccase and a zinc-dependent dehydrogenase) upon incubation, either under drastic (high temperature) or operational conditions. Those two enzymes were co-immobilised reversibly (by electrostatic interactions and His-tag metal coordination) to form a cascade reaction that catalyses the NAD(+)-dependent oxidation of diols coupled to a laccase-mediator for the in situ regeneration of the redox cofactor. Both the protein scaffolds and the metal cofactors undergo rearrangements during operational use or thermal incubation, but they seem to move as a whole unit within the support. Migration inside the support apparently causes only small alterations to the structure of the protein, yet it leads to the exhaustion of the heterogeneous biocatalyst. As such, we show that the use of advanced X-ray spectroscopy with spatial resolution can help obtain a better understanding of the molecular phenomena that occur during the operation of heterogeneous biocatalysts. Overall, this is fundamental to guide the optimisation for more productive and robust bioprocesses based on immobilised enzyme systems.