Purification of Nitrate Reductase from a Hyperthermophylic Archaeon Pyrobaculum

The wide spread pollution of ground water with nitrate is an environmental problem and a health hazard. Nitrate/nitrite pollution is specially significant in agricultural regions where nitrate is used as fertilizer and where animal waste seeps into the water supply. The detection and quantification of N03 by conventional techniques suffers from a laborious, time consuming methodology and interference from ions present in environmental samples.

The goal of this project is to purify the nitrate reductase enzyme from an extremely thermophilic Archaeon named Pyrobaculum aerophilum (growth optimum at 100oC) and to develop a biosensor for the detection of nitrate pollution in the environment. P. aerophilum grows heterotrophically on complex substrates such as yeast extract and peptone which serve as electron donor and carbon source. Either nitrate or oxygen are used as the electron acceptor. Under strict anaerobic growth conditions P. aerophilum reduces nitrate through the denitrification pathway.

P. aerophilum is one of the few hyperthermophilic microorganisms that is capable of utilizing nitrate as electron acceptor. The purified nitrate reductase enzyme can be used for sensing the level of nitrate contamination in water. This will be achieved by electronically joining the nitrate consuming enzyme to a gold electrode. This electrode provides the electrons necessary for the electroenzymatic reduction of nitrate to nitrite. The basis of this technology is that the level of nitrate in environmental samples is proportional to the electrical current that flows from the electrode to the thermostable enzyme. Unlike enzymes from mesophilic microbes which have to be stored at 4oC, the nitrate reductase from the extremely thermophilic P. aerophilum, is expected to remain stable at room temperature. This would greatly enhance the shelf life of the sensor electrode. The thermostability of the nitrate reductase from P. aerophilum would also facilitate the portability of the biosensor to the field without the need of refrigeration. In addition, the nitrate biosensor will be designed for a small sample volume. This will reduce cost and expedite sampling and monitoring of ground water.

Dr. Imke Schroeder, Research Advisor; Sepideh Afshar, Research Assistant