eng Science and Education Publishing International Journal of Environmental Bioremediation & Biodegradation 2333-8636 2018-07-10 6 1 8 17 10.12691/ijebb-6-1-2 IJEBB2018612 article Damien N. McCarthy mccarthyd001@gmail.com 1 Grant C. Edwards 1 Department of Environmental Science, Faculty of Science and Engineering, Macquarie University, Sydney, Australia 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 merB gene, and a sequence with highly conserved active site homology was obtained from E.coli plasmid R8361b. The SBP plus merB 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 E.coli (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. http://pubs.sciepub.com/ijebb/6/1/2/ijebb-6-1-2.pdf merB methylmercury enzyme immobilization demthylation