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Patwardhan, S.V., Emami, F.S., Berry, R.J., Jones, S.E., Naik, R.R., Deschaume, O., Heinz, H. and Perry, C.C., 2012. Chemistry of aqueous silica nanoparticle surfaces and the mechanism of selective peptide adsorption. Journal of the American Chemical Society, 134(14), pp.6244-6256.

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Article

Immobilized Organo-Mercurial Lyase on Zeolite Using a Solid Binding Peptide

1Department of Environmental Science, Faculty of Science and Engineering, Macquarie University, Sydney, Australia


International Journal of Environmental Bioremediation & Biodegradation. 2018, Vol. 6 No. 1, 8-17
DOI: 10.12691/ijebb-6-1-2
Copyright © 2018 Science and Education Publishing

Cite this paper:
Damien N. McCarthy, Grant C. Edwards. Immobilized Organo-Mercurial Lyase on Zeolite Using a Solid Binding Peptide. International Journal of Environmental Bioremediation & Biodegradation. 2018; 6(1):8-17. doi: 10.12691/ijebb-6-1-2.

Correspondence to: Damien  N. McCarthy, Department of Environmental Science, Faculty of Science and Engineering, Macquarie University, Sydney, Australia. Email: mccarthyd001@gmail.com

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 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.

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