Recombinant expression of organophosphorus acid anhydrolase in bacteria : towards environmental biosensors

Abstract

The extensive use of organophosphorus compounds (OPs) in pesticides in order to increase the crop production has led to the accumulation of these highly toxic chemicals in soil water, and food chain. Also the terrorist events happening worldwide lead to the proliferation of the hazardous chemical agents like VX, VR (nerve agents) and chemical contamination outbreak in the food supply. OPs are the most commonly used insecticides and pesticides, which cause a wide range of long-lasting and life-threatening conditions. Due to the acute toxicity and long-term side effects of OP compounds, their timely, on-the-spot and rapid detection has gained importance, for the efficient healthcare management. In this respect, several OP degrading enzymes have gained the spotlight in developing the enzyme-based biosensors, owing to their high activity and broad specificity. Among these microbial enzymes Organophosphorus acid anhydrolase (OPAA) is able to hydrolyze the variety of bonds, like P-F, P-O, P-S and P-CN. OPAA is able to hydrolyze nerve agents and OPs which prompted our interest of using it as the potent agent which can be used for designing the enzyme based biosensor. There have been very few reported works of OPAA activity on paraoxon and methyl parathion and our work shows the increased activity of variant of OPAA (FL) on paraoxon than the previously reported works. In the present piece of work variant of OPAA is expressed, purified and tested for its activity against paraoxon. OPAA showed its optimum activity at pH 8.5 and 50° C. The purified enzyme is further immobilized into the alginate beads for the detection of paraoxon. Furthermore, to improve the detection limits, and sensitivity, free OPAA as well as beads of OPAA and alginate are employed in conjunction with FITC. Keywords: Organophosphorus acid anhydrolase (OPAA), Paraoxon, Acetylcholinesterase (AChE), Chemical warfare nerve agents, Biosensor