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

Damien N. McCarthy^1, and Grant C. Edwards¹

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

Cite this paper:
Damien N. McCarthy and 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

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.

Keywords:
merB methylmercury enzyme immobilization demthylation

This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Figures

References:

[1]	Frohne, T., Rinklebe, J., Langer, U., Du Laing, G., Mothes, S. and Wennrich, R., 2012. Biogeochemical factors affecting mercury methylation rate in two contaminated floodplain soils. Biogeosciences, 9(1), pp.493-507.
[2]	Gilmour, C.C., Henry, E.A. and Mitchell, R., 1992. Sulfate stimulation of mercury methylation in freshwater sediments. Environmental Science & Technology, 26(11), pp.2281-2287.
[3]	Kerin, E.J., Gilmour, C.C., Roden, E., Suzuki, M.T., Coates, J.D. and Mason, R.P., 2006. Mercury methylation by dissimilatory iron-reducing bacteria. Applied and environmental microbiology, 72(12), pp.7919-7921.
[4]	Morel, F.M., Kraepiel, A.M. and Amyot, M., 1998. The chemical cycle and bioaccumulation of mercury. Annual review of ecology and systematics, pp.543-566.
[5]	Lyyra, S., Meagher, R.B., Kim, T., Heaton, A., Montello, P., Balish, R.S. and Merkle, S.A., 2007. Coupling two mercury resistance genes in Eastern cottonwood enhances the processing of organomercury. Plant biotechnology journal, 5(2), pp.254-262.
[6]	Basak, B. and Dey, A., 2015. Bioremediation Approaches for Recalcitrant Pollutants: Potentiality, Successes and Limitation. Toxicity and Waste Management Using Bioremediation, p.178.
[7]	Boyd, E. and Barkay, T., 2012. The mercury resistance operon: from an origin in a geothermal environment to an efficient detoxification machine.Frontiers in microbiology, 3, p.349.
[8]	Fox, B. and Walsh, C.T., 1982. Mercuric reductase. Purification and characterization of a transposon-encoded flavoprotein containing an oxidation-reduction-active disulfide. Journal of Biological Chemistry, 257(5), pp.2498-2503.
[9]	Parks, J.M., Guo, H., Momany, C., Liang, L., Miller, S.M., Summers, A.O. and Smith, J.C., 2009. Mechanism of Hg− C Protonolysis in the Organomercurial Lyase MerB. Journal of the American Chemical Society, 131(37), pp.13278-13285.
[10]	Silva, P.J. and Rodrigues, V., 2015. Mechanistic pathways of mercury removal from the organomercurial lyase active site. PeerJ, 3, pp 1127.
[11]	Lafrance-Vanasse, J., Lefebvre, M., Di Lello, P., Sygusch, J. and Omichinski, J.G., 2009. Crystal structures of the organomercurial lyase merb in its free and mercury-bound forms insights into the mechanism of methylmercury degradation. Journal of Biological Chemistry, 284(2), pp.938-944.
[12]	Di Lello, P., Benison, G.C., Valafar, H., Pitts, K.E., Summers, A.O., Legault, P. and Omichinski, J.G., 2004. NMR structural studies reveal a novel protein fold for MerB, the organomercurial lyase involved in the bacterial mercury resistance system. Biochemistry, 43(26), pp.8322-8332.
[13]	Boopathy, R., 2000. Factors limiting bioremediation technologies. Bioresource technology, 74(1), pp.63-67.
[14]	Wingard, L.B., Katchalski-Katzir, E. and Goldstein, L. eds., 2014.Immobilized enzyme principles: applied biochemistry and bioengineering (Vol. 1). Elsevier.
[15]	Care, A., Bergquist, P.L. and Sunna, A., 2015. Solid-binding peptides: smart tools for nanobiotechnology. Trends in biotechnology, 33(5), pp.259-268.
[16]	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.
[17]	Sunna, A., Chi, F. and Bergquist, P.L., 2013. A linker peptide with high affinity towards silica-containing materials. New biotechnology, 30(5), pp.485-492.
[18]	Murtaza, I., Dutt, A. and Ali, A., 2002. Biomolecular Engineering of Escherichia coli Organo-mercurial Lyase Gene and its Expression, Indian Jnl of Biotech, Vol. 1, pp 117-120.
[19]	Mertens, N., Remaut, E. and Fiers, W., 1995. Tight transcriptional control mechanism ensures stable high-level expression from T7 promoter-based expression plasmids. Nature Biotechnology, 13(2), pp.175-179.
[20]	Waugh, D.S., 2005. Making the most of affinity tags. Trends in biotechnology, 23(6), pp.316-320.
[21]	Nygaard, S., Wendelbo, R. and Brown, S., 2002. Surface‐specific zeolite‐binding proteins. Advanced Materials, 14(24), pp. 1853-1856.
[22]	Prilusky, J., Felder, C.E., Zeev-Ben-Mordehai, T., Rydberg, E.H., Man, O., Beckmann, J.S., Silman, I. and Sussman, J.L., 2005. FoldIndex©: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics, 21(16), pp.3435-3438.