One-Step Purification and Immobilization of Recombinant Enzymes Using a Bifunctional Epoxy Support: A Case Study with Phenylalanine Ammonia-Lyase and Transaminase

Abstract

This work investigates efficient immobilization strategy for recombinant biocatalysts which does not require preliminary enzyme purification, thereby avoiding the costly and time-consuming downstream processes associated with traditional chromatographic isolation. Our approach utilizes a macroporous poly(methyl methacrylate) resin as a solid carrier decorated with mixed functions of both coordinative binding ability (via metal ion chelation for affinity capture) and covalent bond-forming ability (via epoxide groups for stabilization). This heterofunctional surface enabled the single-operation enrichment and immobilization of two structurally and functionally distinct enzymes: the homotetrameric phenylalanine ammonia-lyase and the dimeric, pyridoxal-5'-phosphate-dependent transaminase. The results demonstrate that a specific surface architecture—characterized by a rigid trisepoxide linker and a defined complexing group-to-epoxy ratio—proved to be optimal for the two diverse histidine-tagged enzymes, governed primarily by the physicochemical properties of the carrier.