Upgrading Epoxy Supports for Enzyme Immobilization by Affinity Function Doping—A Case Study with Phenylalanine Ammonia-Lyase from Petroselinum crispum

• Published in: Catalysts Vol. 14; no. 1; p. 14
• Main Authors: Alács, Bálint, Zrinyi, Anna, Hornyánszky, Gábor, Poppe, László, Bell, Evelin
• Format: Journal Article
• Language: English
• Published: Basel MDPI AG 01.01.2024
• Subjects: Acids; Affinity; Ammonia; Binding; Biocatalysts; Case studies; Cobalt; Covalence; Covalent bonds; Diamines; Doping; E coli; Enzymes; Epoxy resins; Immobilization; Nanoparticles; Phenylalanine; Proteins
• ISSN: 2073-4344; 2073-4344
• DOI: 10.3390/catal14010014

This article provides a method to upgrade epoxy-functionalized carriers for covalent enzyme immobilization to selective carriers suitable for covalent immobilization of metal affinity-tagged enzymes without the need of preliminary enzyme purification. Affinity function doping of the epoxy-functionalized surface introduces an advanced possibility to avoid the costly and time-consuming downstream processes required for efficient immobilization on non-selective epoxy carriers. Our approach is based on the partial functionalization of surface epoxides via a proper diamine-derived linker and an ethylenediaminetetraacetic dianhydride-based chelator charged with cobalt ions. The solid macroporous carriers, doped with metal affinity functions, have both coordinative binding ability (rapid anchoring the metal affinity-tagged enzymes to the surface) and subsequent covalent bond-forming ability (preferred binding of the tagged enzyme to the surface after proper washing by the residual epoxide functions), enabling a single operation for the enrichment and immobilization of a recombinant phenylalanine ammonia-lyase from parsley fused to a polyhistidine affinity tag. The immobilized PcPAL was applied in the ammonia elimination of racemic phenylalanine, 4-chlorophenylalanine, and 4-bromophenylalanine to produce the corresponding d-phenylalanines, in addition to the formation of (E)-cinnamates, as well as in ammonia addition reactions to (E)-cinnamates, yielding the corresponding enantiopure l-phenylalanines.