Self-assembly of amyloid fibrils that display active enzymes

Enzyme immobilization is an important strategy to enhance the stability and recoverability of enzymes and to facilitate the separation of enzymes from reaction products. However, enzyme purification followed by separate chemical steps to allow immobilization on a solid support reduces the efficiency and yield of the active enzyme. Here we describe polypeptide constructs that self-assemble spontaneously into nanofibrils with fused active enzyme subunits displayed on the amyloid fibril surface. We measured the steady-state kinetic parameters for the appended enzymes in situ within fibrils and compare these with the identical protein constructs in solution. Finally, we demonstrated that the fibrils can be recycled and reused in functional assays both in conventional batch processes and in a continuous-flow microreactor. Self-immobilizing enzymes: Fusion of a prion domain and a globular enzyme allows spontaneous assembly of fibrils to form an amyloid core decorated with active enzymes. Comparison of steady-state enzyme kinetic parameters for soluble and fibrillar forms of the fusion proteins indicates how immobilization affects substrate binding and catalytic turnover. The amyloid-immobilized enzymes can be recycled and reused in both batch mode and continuous flow.