Application of Surface Expresses Phosphotriesterase for Detoxification and Monitoring of Organophosphorus PesticidesEPA Grant Number: R823663
Title: Application of Surface Expresses Phosphotriesterase for Detoxification and Monitoring of Organophosphorus Pesticides
Investigators: Mulchandani, Ashok , Chen, Wilfred
Institution: University of California - Riverside
EPA Project Officer: Lasat, Mitch
Project Period: October 1, 1995 through September 1, 1998 (Extended to September 30, 1999)
Project Amount: $311,766
RFA: Exploratory Research - Engineering (1995) Recipients Lists
Research Category: Land and Waste Management , Engineering and Environmental Chemistry
At the time when the public concern about the organophosphorous pesticide residues in food, water and the environment is increasing, the use of these pesticides in agriculture has also increased. Discovery and development of novel biological treatments will enable the design for alternative biodegradation processes in place of conventional techniques. Phosphotriesterase isolated from Pseudomonas and Flavobacterium has been shown to degrade organophosphates, and immobilized phosphotriesterase has been applied as a means to detoxify pesticide wastes. However, construction of an enzyme reactor is often very labor intensive and could suffer from diffusional limitations. The proposed research will seek to develop a novel method for biodegradation of organophosphorus pesticide in an immobilized cell bioreactor using E. coli with surface-exposed phosphotriesterase as "live" biocatalysts and to couple the reactor with a flow injection system for the detection of organophosphorus pesticides. Parathion, being the most frequently employed organophosphate pesticide in the US, will be used to demonstrate the potential of this new method.
A lpp-ompA-opd fusion will be used to direct phosphotriesterase onto E. coli surface. The effect of induction, temperature, and cofactor concentration, all implicated in influencing the extent of surface-exposure, will be studied. Responses of plasmid stability, cell growth, and degradation kinetics to various operating conditions such as oxygen tension, parathion and methanol concentration, selection of expression hosts, and immobilization will be monitored and applied to define an optimum reactor condition. Large-scale detoxification of parathion will be investigated in an immobilized cell bioreactor with the recombinant cells immobilized on a solid support. Additional experiments to further optimize the bioreactor operation will concentrate on the ability to stabilize enzyme activity and the ability to regenerate the reactor activity. Novel biosensors based on the monitoring of p-nitrophenol or other phenolic compounds, formed during the hydrolysis of parathion or other organophosphate pesticide by the whole cells surface expressing phosphotriesterase, optically and amperometrically will be developed for the quantitative determination of parathion and other organophosphorus pesticides. These studies will provide a thorough investigation of a potentially reliable and economical means to detoxify and monitor pesticides and nerve agents using "live" immobilized biocatalysts.