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 (YA=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.
Highlights
The expansion of employing enzymes in food and pharmaceutical industry, development of novel biosensors and synthesis of small organic compounds spawned the need of creating more stable and recyclable biocatalysts [1,2,3]
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
To find an appropriate metal ion for the IMAC-type immobilization, the different forms of His-tagged PcPAL anchored to EDTA-based IMAC support charged with transition metal ions–iron(III), cobalt(II), nickel(II), copper(II), zinc(II)– were characterized by thermal stability and reusability tests
Summary
The expansion of employing enzymes in food and pharmaceutical industry, development of novel biosensors and synthesis of small organic compounds spawned the need of creating more stable and recyclable biocatalysts [1,2,3]. The appropriate immobilization method and a suitable carrier for it can significantly improve the properties of the biocatalyst [26,27,28]. In the late 1970s, Porath and coworkers discovered that histidine, cysteine and tryptophan amino acids can coordinate with transitional metal ions [29, 30]. This phenomenon called immobilized metal ion affinity chromatography (IMAC), is used consciously for protein purification [31]. Histidine tags artificially attached to the recombinant target proteins make them suitable to form stable metal-protein complexes, facilitating a simple and effective way for protein purification
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