Overexpression and crosslinking of the metallo-chaperone PbrD to calcium alginate nanoparticles as a novel biosorbent for lead ions

Abstract

Heavy metals are one of the most harmful pollutants found in our fresh water resources; threatening human health, infrastructure and the entire eco-system. Amongst other metals present in water, lead (Pb) has no significance in all forms of life and is toxic even at low concentrations. Despite several reviews on Pb toxicity, the remediation of Pb ions [Pb(II)] from water systems is scarce and understudied. This project aimed to design a novel in vitro Pb-nanobiosorbent utilizing a bacterial metallo-chaperone protein PbrD derived from Cupriavidus metallidurans CH34. Genetic engineering and molecular techniques were applied to obtain a purified refolded PbrD fusion protein (rPbrD) from E. coli; the first for the prokaryotic Pb metallo-chaperone. Western blot and peptide sequencing confirmed the identity of the recombinant rPbrD. Bioinformatics and circular dichroism spectra of rPbrD indicated an arrangement of α-helices, antiparallel β-sheets (Trxtag), turns and loops which was significantly altered in the presence of Pb(II). Tryptophan fluorescence of rPbrD showed significant fluorophore quenching in the presence of Pb(II) suggesting intermolecular interactions with an appropriately folded rPbrD protein. The refolded fusion protein was cross-linked to calcium alginate nanoparticles (rPbrD-CANPs) for its intended use as a biosorbent. Scanning and transmission electron microscopy (SEM, TEM) coupled with energy dispersive X-ray spectroscopy (EDS), dynamic light scattering (DLS) and zeta potential analysis revealed uniformly synthesized, mono-dispersed nanoparticles displaying a negative surface charge indicative of a stable colloidal system allowing interactions with Pb(II). Binding activity of rPbrD, rPbrD-CANPs and their respective controls were analyzed by quantifying metal uptake using inductively coupled plasma mass spectroscopy (ICP-MS). At the highest concentration of Pb tested (1 mg/L) rPbrD biosorbed 99.7 % at a rate of 64 μg/g. Additionally, surface cross-linked rPbrDCANPs bisorbed 99.9 % of 100 mg/L Pb(II) at a rate of 8820 μg/g. The Pb binding ability by rPbrD and rPbrD-CANPS was indicative of its maintained functionality even at elevated concentrations of Pb(II). These results suggest that rPbrD-CANPs could be applied in the remediation of Pb-contaminated wastewater. As such, treated wastewater could be recycled for water use in an era where water scarcity is at the forefront of global threats.