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Nitrogen Oxy-Anions : Optical Biosensing in the Environment

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Summary: The metalloprotein cytochrome cd1 nitrite reductase has been extracted and purified from the bacterium Paracoccus denitrificans (formerly Thiosphaera pantrotropha). The bacterium was cultured in a 200 I bioreactor and was harvested using a cross-flow filtration system. The bacterium was fractionated and the soluble nitrite reductase enzyme was purified from the periplasmic fraction using a combination of anion-exchange chromatography, ammonium sulphate precipitation and nickel affinity chromatography. The final yield of the cytochrome cd1 nitrite reductase was 1g. Solutions studies have shown that this enzyme specifically interacts with nitrite ions even in the presence of high concentrations of other potentially interfering ions. The interaction of the nitrite anion with the enzyme is indicated by changes in both the spectroscopic and redox properties of the haem centre of the protein. These changes can be related directly to the concentration of the nitrite ions present in the solution.
Summary: The metalloprotein nitrate reductase has been extracted and purified from a genetically modified strain of the bacterium Paracocus denitrificans (formerly Thiosphaera pantrotropha). The bacterium was cultured in a 200 I bioreactor and was harvested using a cross-flow filtration system. The bacterium was fractionated and the soluble nitrate reductase enzyme was purified from the periplasmic fraction using a combination of anion-exchange chromatography, ammonium sulphate precipitation and nickel affinity chromatography. The final yield of the nitrate reductase was ca. 30mg. Solution studies have shown that this enzyme specifically interacts with nitrate ions even in the presence of high concentrations of other potentially interfering ions. The interaction of the nitrate anion with the enzyme is indicated by changes in both the spectroscopic and redox properties of the haem centre of the protein. These changes can be related to the concentration of the nitrate ions present in the solution.
Summary: Using the metalloprotein cytochrome cd1 nitrite reductase as the molecular recognition element, the partners have developed a biosensing system for the detection of nitrite ions in environmental waters. The nitrite reductase has been encapsulated in a silica sol-gel matrix. The sol-gel protects the enzyme - extending the lifetime of the sensing device - whilst allowing the target analyte (nitrite ions) to diffuse and interact with the nitrite reductase. Upon interaction with the nitrite ions there is a consequent change in the UV-visible absorption spectrum of the nitrite reductase. The change of the absorption spectrum has been shown to be directly proportional to the concentration of the nitrite ions present in the water sample. Calibration has been achieved at 3 different wavelengths in the concentration range suitable for use in potable and other environmental water samples. The sensor has been tested against interferent species and shows excellent specificity. The developed biosensor is based on optical measurements. It should be noted that as the enzyme contains a redox centre the device could be used to quantify concentrations of nitrite ions via electrochemical measurements.

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