Immobilization of Phenylalanine Ammonia-lyase via EDTA Based Metal Chelate Complexes – Optimization and Prospects

Abstract

Immobilized metal ion affinity chromatography principles were applied for selective immobilization of recombinant polyhistidine tag fused phenylalanine ammonia-lyase from parsley (PcPAL) on porous polymeric support with aminoalkyl moieties modified with an EDTA dianhydride (EDTADa)-derived chelator and charged with transition metal ions. Out of the five investigated metal ions - Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺ - the best biocatalytic activity of PcPAL was achieved when the enzyme was immobilized on the Co²⁺ ion-charged support (31.8 ± 1.2 U/g). To explore the features this PcPAL obtained by selective immobilization, the thermostability and reusability of this PAL biocatalyst were investigated. To maximize the activity of the immobilized PcPAL the surface functionalization of the aminoalkylated polymeric carrier was fine-tuned with using glycidol as a thinning group beside EDTADa. The maximal activity yield (Y_A=103 %) was earned when the EDTADa and glycidol were used in 1 to 24 ratio. The reversibility of the immobilization method allowed the development of a support regeneration protocol which enables easy reuse of the functionalized support in case of enzyme inactivation.