@article{ijebb2018612,
author={{McCarthy, Damien N. and Edwards, Grant C.},
title={Immobilized Organo-Mercurial Lyase on Zeolite Using a Solid Binding Peptide},
journal={International Journal of Environmental Bioremediation & Biodegradation},
volume={6},
number={1},
pages={8--17},
year={2018},
url={http://pubs.sciepub.com/ijebb/6/1/2},
issn={2333-8636},
abstract={Methylmercury (MeHg) compounds can form naturally, are highly toxic, and of concern because of their tendency to bio-accumulate. Certain bacteria have evolved mechanisms that can tolerate MeHg by first demethylating MeHg compounds, before further processing. Drawing inspiration from this demethylation mechanism controlled by a single organo-mercurial lyase in a protonolysis reaction, this research uses a recombinant gene that produces this lyase plus an additional polypeptide that selectively binds to zeolite particles, effectively tethering the enzyme to the solid substrate. This work is part of a broader attempt to create a fixed bed reactor for de-methylation of MeHg. Enzyme immobilization was achieved using a solid binding peptide (SBP) with high affinity for faujasite zeolite (FZ), the choice of binding substrate in the present work. The lyase is coded for by the <i>merB </i>gene, and a sequence with highly conserved active site homology was obtained from <i>E.coli</i> plasmid R8361b. The SBP plus <i>merB</i> sequence was designed such that the SBP was positioned either on the N or C-terminal of the construct. The DNA was synthesized commercially, and expressed in <i>E.coli</i> (BL21DE3 Star) using pET100? vector. Sanger sequencing was used to confirm construct in transformed cells using standard T7 oligos. Expression was lactose induced, and SDS-PAGE electrophoresis was used to confirm protein production and size. LC-MS/MS and sequence bio-analytics confirmed peptide sequence. Silica binding assays using SDS-PAGE confirmed binding of the enzyme to the silica substrate. Enzyme functionality results using a non-methylated mercuric compound were inconclusive, however the enzyme has not been assessed using MeHg compounds at this stage.},
doi={10.12691/ijebb-6-1-2}
publisher={Science and Education Publishing}
}