Recent security and environmental concerns have resulted in a plethora of research into the development of novel sensors for explosives and drugs. The high specificity of enzymes which interact with one or a range of specific substrates and catalyse only one type of chemical reaction, plus the possibility to use such enzymes to catalyse a specific biochemical reaction outside the cell has opened up a multitude of possibilities for the use of enzymes in analytical applications.
This work demonstrates biological recognition elements for both an explosive
and drug biosensor based on the isolation and characterization of bacterial enzymes namely nitroreductases, due to their ability to degrade many commonly used explosives and carboxylesterases, because they have the potential to hydrolyse cocaine into its main metabolites. The enzymes have been genetically modified to incorporate the cys-tag (6 cysteine amino acids) at the enzyme N-terminus to enable the enzyme to adhere to the biosensor electrode surface (gold surface), subsequently the effect of cys-tag insertion on enzymes molecular (gene isolation, cloning, expression, and purification) and biochemical characterization (protein concentration, specific activity, K_m, V_max, pH and temperature), and observe the reaction pathways of the enzymes.
AIl the bacterial Nitroreductases (NfnB, YdjA, Dde_0086, and Dde_2199)
showed considerable activity with different nitroaromatic compounds and displayed different rates of reaction for each substrate.
The optimum pH and temperature were evaluated along with their K_m and V_max
values. Additionally bacterial carboxylesterases (PnbA1 and PnbA2) were found to be active with cocaine, and hydrolysed cocaine into benzoylecgonine and methanol rather than ecgonine methyl ester and benzoic acid following human liver carboxylesterase 1(hCE-1) in its pathway to hydrolyse cocaine.
The cys-tag insertion revealed its effect through decrease enzyme specific activity, protein concentration, K_m and V_max values, optimum temperature range for enzyme stability, however it does not restrict substrate entrance into the active site and all the bacterial nitroreductases and carboxylesterases remain active, furthermore it does not affect the optimum pH for enzyme activity.