Warelow, TP; (2015) Arsenite oxidase as a novel biosensor for arsenite. Doctoral thesis , UCL (University College London).

Abstract

Contamination of groundwater with the toxic soluble arsenic species, arsenite (AsIII) and arsenate (AsV) has led to an epidemic of arsenic poisoning effecting over 100 million people worldwide. The World Health Organisation (WHO) recommended maximum contaminant level (MCL) for arsenic in water is 0.13 μM (10 μg L−1). Accurate quantification of arsenic below the MCL usually requires highly sensitive laboratory based techniques, the practical uses of which are limited within the effected populations, principally due to cost. Biosensors are a potentially powerful technology for overcoming this problem. Amperometric biosensors couple the analytical sensitivity of electrochemistry with the selectivity of enzyme substrate interactions. The bioenergetic metalloenzyme AsIII oxidase (Aio) catalyses the oxidation of AsIII to AsV in a number of physiologically diverse microorganisms including the Rhizobium sp. str. NT-26. To develop a biosensor for AsIII it was first necessary to optimise the expression and purification of the biological recognition element, a recombinant NT-26 Aio in Escherichia coli str. DH5α. with final a yield of ca. 1.1 mg per L of culture. The recombinant NT-26 Aio was characterised using biophysical techniques to confirm the correct insertion of the enzyme cofactors during heterologous expression in E. coli. The reduction midpoint potentials of the 3Fe-4S (270 mV) and the Rieske 2Fe-2S (225 mV) clusters were confirmed by redox titration. The thermostability of the recombinant Aio was ≤ 64.5 °C. The oxidised structure of the Mo at the active site was confirmed to have a di-oxo (Mo = O2) coordination. The kinetics and pH dependence of AsIII oxidation were investigated using various artificial and physiological electron acceptors. Electrochemical studies were performed to develop a system for AsIII concentration determination, using the biological recognition element Aio. The electron transfer mediator ferrocene methanol was found to produce the greatest currents during catalytic voltammetry experiments at pH 8.0. A chronoamperometric detection system incorporating the electron transfer mediators ferrocene methanol and potassium ferricyanide was able to resolve AsIII concentrations of 0.07 – 0.53 μM (5 – 40 μg L−1), below the WHO MCL for arsenic, suggesting such a system would be capable of determining the safe levels of arsenic in drinking water.

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