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Bioengineering a thermo-stable oxyanion reductase for enhanced selenate bioremediation

Reference	BB/D00781X/2
Principal Investigator / Supervisor	Dr Clive Butler
Co-Investigators / Co-Supervisors	Dr Richard Lewis, Professor David Richardson
Institution	University of Exeter
Department	Biosciences
Funding type	Research
Value (£)	295,116
Status	Completed
Type	Research Grant
Start date	02/01/2007
End date	01/07/2010
Duration	42 months

Abstract

Selenium (Se) is a naturally occurring trace element in low abundance. It is essential for humans and animals but in its most oxidised form, selenate, is highly soluble and can present a significant hazard to health and the environment. High levels of selenate are found naturally in a limited number of areas worldwide but it is more commonly human activities that significantly contribute to generating selenate contaminated environments. One of the most successful methods for selenate detoxification is via biotic reduction of selenate to selenite followed by either biotic or abiotic reduction of selenite to insoluble less-toxic elemental selenium. The only selenate reductases characterised so far are from mesophilic organisms and examples of both soluble and membrane-bound enzymes have been reported. By far the most comprehensively studied example is the periplasmic selenate reductase (SerABC) from T. selenatis. We have identified an unusual nitrate reductase gene cluster (AF0175-6) in the hyperthermophilic archaeon, A. fulgidus, which encodes an enzyme system that displays high homology to SerAB. Using the recent crystal structures of the respiratory membrane bound nitrate reductase (NarG) from E. coli we have modelled the active site of SerA and AF0176, and identified a number of key residues that we suggest might control substrate selectivity. During this project a combination of molecular modelling and site directed mutagenesis will be used to modify key amino acid residues around the active site, altering substrate selectivity of AF0176 towards selenate rather than nitrate. This project has the long-term objective of bioengineering a thermo-stable selenate reductase for use in an enzyme based thermo-extraction bioremediation strategy.

Summary

Selenium is a naturally occurring chemical element. It is important for the health of both humans and animals, but can also be very toxic at high concentrations. Selenium can be found in a number of different chemical forms. In the presence of oxygen, it is usually in a compound called selenate. Selenate is highly soluble in hot water, highly toxic, and when released into the environment can present a significant hazard to health. Selenium contamination usually occurs due to the discharge of selenate waste from heavy industries such as glass production, copper smelting and fossil fuel combustion. One of the most successful ways to clean selenate contaminated areas is by the use of micro-organisms in a process called bioremediation. Micro-organisms can detoxify selenate by chemically modifying the compound back to the elemental form, selenium. Elemental selenium is far less toxic than selenate and is insoluble, forming a red solid substance that can be easily separated from the contaminated soil or water. Some micro-organisms carry out this chemical modification of selenate using a specialised reactive molecule or enzyme called selenate reductase. During a recent study of the genetic sequences of a large number of micro-organisms we have identified a sequence (gene) in an ancient micro-organism called A. fulgidus that codes for an enzyme that has a very similar structure to selenate reductase. This organism lives in deep sea hot-water vents (hydrothermal vents) at temperatures near 95oC. Comparing the sequence of the A. fulgidus enzyme with those from other micro-organisms we have highlighted regions in the sequence of the selenate reductase that may be responsible for why the enzyme reacts with selenate and not other similar compounds such as nitrate. By changing the sequence of the A. fulgidus enzyme we hope to make it behave more like the selenate reductase enzyme, but also work at high temperatures. The advantage of this work is that if we can engineer a selenatereductase enzyme that works at high temperature we can use it to remove a higher concentration of selenate from contaminated environments.

Committee	Closed Committee - Engineering & Biological Systems (EBS)
Research Topics	Industrial Biotechnology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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