Grantee Research Project Results
Final Report: Application of Surface Expresses Phosphotriesterase for Detoxification and Monitoring of Organophosphorus Pesticides
EPA Grant Number: R823663Title: 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: Aja, Hayley
Project Period: October 1, 1995 through September 1, 1998 (Extended to September 30, 1999)
Project Amount: $311,766
RFA: Exploratory Research - Engineering (1995) RFA Text | Recipients Lists
Research Category: Land and Waste Management , Safer Chemicals
Objective:
While public concern about the organophosphorus pesticide residues in food, water, and the environment is increasing, the use of these pesticides in agriculture also has 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 diffusional limitations may be encountered. The proposed research will seek to develop a novel method for the 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, the most frequently employed organophosphate pesticides in the United States, will be used to demonstrate the potential of this new method.Summary/Accomplishments (Outputs/Outcomes):
Phosphotriesterase, also known as organophosphorus hydrolase (OPH), was displayed and anchored onto the surface of E. coli using an Lpp-OmpA fusion system with a tightly regulated tac promoter. Production of the fusion protein was verified by immunoblotting with OmpA antisera. Inclusion of the organophosphorus hydrolase signal sequence was necessary for achieving enzymatic activity on the surface. More than 80 percent of the OPH activity was located in the cell surface, as determined by protease accessibility. Whole cells expressing OPH on the cell surface degraded parathion and paraoxon very effectively without the diffusional limitation, resulting in seven-fold higher rates of parathion degradation compared to the whole cells with similar levels of intracellular OPH.OPH activity on the cell surface was found to be a function of several factors. It was highly host-specific; a high rate of parathion degradation was observed from strain JM105 and XL1-Blue, which regulated production of OPH fusion very tightly. OPH activity was highly dependent on growth conditions and growth medium. Optimum activity was observed when the cells were grown in Luria-Bertani-buffered medium at 37 C supplemented with cobalt chloride, which favors the formation of a metal-active center. The timing of cobalt addition also influenced enzyme activity, with the maximum activity resulting by the addition of cobalt to the induced cultures during the late stationary phase. Under optimum growth conditions, cultures had an 8-fold higher parathion degradation rate.
The cells expressing OPH on the cell surface were subsequently immobilized on support matrix and used for the degradation of organophosphate compounds. Two methods of cell immobilization were used. In the first method, cells were immobilized on the surface of nonwoven polypropylene fabric. The best cell loading (256 mg cell dry weight per g of support) and subsequent hydrolysis of organophosphate nerve agents were achieved by immobilizing non-growing cells from a pH 8, 50-mM citrate-phosphate buffer supplemented with 1 mM Co2+ for 48 h via simple adsorption, followed by organophosphate hydrolysis in a similar buffer supplemented with 0.05-mM Co2+ and 20 percent methanol at 37oC. In batch operations, the immobilized cells degraded 100 percent of a model organophosphate nerve agent, paraoxon, in less than 2 h. The immobilized cells retained almost 100 percent activity during the initial 6 repeated cycles and close to 90 percent activity even after 12 repeated cycles, extending over a period of 19 days without any nutrient supplementation. In addition to paraoxon, other commonly used organophosphates, such as diazinon, coumaphos, and methyl parathion were hydrolyzed efficiently. The cell immobilization technology developed here paves the way for an efficient, simple, and cost-effective method for the detoxification of organophosphate nerve agents.
In the second method, cells were immobilized, post culturing, in pores and the surface of porous glass beads (2-3 mm diameter, 60-300 µm pore size, 55-60 percent pore volume and 24,440 ft2/ft3 surface area) by adsorption followed by cross-linking, and then used in a column (2.5 cm diameter and 9 cm long) as a packed bed reactor for the degradation of paraoxon and coumaphos in a continuous flow-through reactor. The immobilized cells degraded more than 98 percent of 0.1 mM paraoxon up to a flow rate of 250 ml/h, and 96 percent of 1 mM paraoxon up to a flow rate of 175 ml/h. The degradation of coumaphos by these immobilized cells was less effective; only 80 percent and 45 percent of 0.1 mM and 0.3 mM, respectively, of the feed was hydrolyzed at a flow rate of 23 ml/h. The immobilized reactor was very stable and retained over 90 percent of its original activity for over a 3-month period when stored at room temperature in a citrate-phosphate, pH 8 buffer.
Potentiometric and optical biosensors based on the cells expressing OPH on the cell surface were developed and evaluated for monitoring organophosphate compounds. The potentiometric microbial biosensor for the direct measurement of organophosphate (OP) nerve agents was developed by modifying a pH electrode with an immobilized layer of Escherichia coli cells expressing OPH on the cell surface. OPH catalyzes the hydrolysis of organophosphorus pesticides to release protons, the concentration of which is proportional to the amount of hydrolyzed substrate. The sensor signal and response time were optimized with respect to the buffer pH, ionic concentration of the buffer, temperature, and weight of cells immobilized using paraoxon as the substrate. The best sensitivity and response time were obtained using a sensor constructed with 2.5 mg of cells and operating in pH 8.5, 1 mM HEPES buffer. Under these conditions, the biosensor was used to measure as low as 2 µM of paraoxon, methyl parathion, and diazinon. The biosensor had very good storage and multiple-use stability. A similar design was also used to construct potentiometric an enzyme-based biosensor by immobilizing purified OPH enzyme on the tip of the pH electrode. Similar selectivity, sensitivity, lower detection limits, and stability were obtained.
An optical biosensor for the direct determination of organophosphorus compounds was developed by immobilizing the cells expressing OPH in low melting point agarose on a nylon membrane and attaching them to the common end of a bifurcated fiber-optic bundle. OPH-expressing E. coli cells catalyze the hydrolysis of organophosphorus pesticides to form stoichiometric amounts of chromophoric products that absorb light at a specific wavelength. The back-scattered radiation of the specific wavelength incident light was measured using a photomultiplier detector and correlated to the organophosphate concentration. The sensor signal and response time were optimized with respect to the buffer pH and the weight of cells immobilized using parathion as the substrate. The best sensitivity and response time were obtained using a sensor constructed with 1.5 mg of cells operating in pH 9, 50 mM HEPES buffer at 30oC. These conditions were then used to measure concentrations of paraoxon, parathion, and coumaphos pesticides. The biosensor had very good storage and multiple-use stability. A similar design also was used to construct an optical enzyme-based biosensor by immobilizing purified OPH enzyme on the tip of the bifurcated fiber bundle by cross-linking with glutaraldehyde. Similar selectivity, sensitivity, lower detection limits, and stability were obtained.
Three different amperometric biosensors based on the organophosphorus hydrolase enzyme have been developed. In the first format, the enzyme was immobilized covalently onto porous glass beads that were packed in a column and placed upstream of an electrochemical detector for flow injection analysis. The electrochemical detector consisted of a thin-channel three electrode cell consisting of a carbon paste working electrode, a silver/silver chloride reference electrode, and a stainless steel counter electrode. The working electrode was poised at 0.9 V versus the reference, the potential determined to be the optimum for electrooxidation of p-nitrophenol. This biosensor was found to be 20-fold more sensitive than the potentiometric and fiber-optic biosensors with the lower detection limit of 0.1 µM paraoxon and methyl parathion. Subsequently, the carbon paste electrode modified with the enzyme and immobilized in Nafion polymer to demonstrate the application of the biosensor for remote in situ monitoring of OP nerve agents. A disposable format of the amperometric biosensor using screen printed electrodes modified with the purified enzyme also has been developed. The latter two formats are suitable for on-field monitoring of the pesticides.
Future plans are to: (1) apply the immobilized cells bioreactor to high concentration coumaphos insecticide containing waste generated in large volume along the Texas-Mexico border; (2) model the immobilized cell bioreactor and apply the model to run simulations, such a model will be useful for process analysis and control; (3) develop portable hand-held enzyme and microbial biosensors that will be useful for onsite monitoring of water from rivers, lakes and wells; and (4) develop protective clothing to be used by pesticide sprayers and emergency response teams.
Journal Articles on this Report : 11 Displayed | Download in RIS Format
| Other project views: | All 22 publications | 11 publications in selected types | All 11 journal articles |
|---|
| Type | Citation | ||
|---|---|---|---|
|
|
Chen W, Mulchandani A. The use of live biocatalysts for pesticide detoxification. Trends in Biotechnology, February 1998;16(2):71-76. |
R823663 (Final) |
not available |
|
|
Kaneva I, Mulchandani A, Chen W. Factors influencing parathion degradation by recombinant Escherichia coli with surface-expressed organophosphorus hydrolase. Biotechnology Progress 1998;14(2):275-278. |
R823663 (Final) |
not available |
|
|
Mulchandani A, Mulchandani P, Kaneva I, Chen W. Biosensor for direct determination of organophosphate nerve agents using recombinant Escherichia coli with surface-expressed organophosphorus hydrolase. 1. Potentiometric microbial electrode. Analytical Chemistry 1998;70(19):4140-4145. |
R823663 (Final) |
not available |
|
|
Mulchandani A, Kaneva I, Chen W. Biosensor for direct determination of organophosphate nerve agents using recombinant Escherichia coli with surface-expressed organophosphorus hydrolase. 2. Fiber optic microbial biosensor. Analytical Chemistry 1998;70(23):5042-5046. |
R823663 (Final) |
not available |
|
|
Mulchandani A, Mulchandani P, Chen W, Wang J, Chen L. Amperometric thick film strip electrodes for monitoring organophosphate nerve agents based on immobilized organophosphorus hydrolase. Analytical Chemistry 1999;71(11):2246-2249. |
R823663 (Final) |
not available |
|
|
Mulchandani A, Kaneva I, Chen W. Detoxification of organophosphate nerve agents by immobilized Escherichia coli with surface-expressed organophosphorus hydrolase. Biotechnology and Bioengineering 1999;63(2):216-223. |
R823663 (Final) |
not available |
|
|
Mulchandani A, Pan ST, Chen W. Fiber-optic enzyme biosensor for direct determination of organophosphate nerve agents. Biotechnology Progress 1999;15(1):130-134. |
R823663 (Final) |
not available |
|
|
Mulchandani P, Mulchandani A, Kaneva I, Chen W. Biosensor for direct determination of organophosphate nerve agents. 1. Potentiometric enzyme electrode. Biosensors & Bioelectronics 1999;14(1):77-85. |
R823663 (Final) |
not available |
|
|
Richins R, Kaneva I, Mulchandani A, Chen W. Biodegradation of organophosphorus pesticides by surface-expressed organophosphorus hydrolase. Nature Biotechnology 1997;15(10):984-987. |
R823663 (Final) |
not available |
|
|
Rogers KR, Wang Y, Mulchandani A, Mulchandani P, Chen W. Organophosphorus hydrolase-based assay for organophosphate pesticides. Biotechnology Progress 1999;15(3):517-521. |
R823663 (Final) |
not available |
|
|
Wang J, Chen L, Mulchandani A, Mulchandani P, Chen W. Remote biosensor for in-situ monitoring of organophosphate nerve agents. Electroanalysis 1999;11(12):866-869. |
R823663 (Final) |
not available |
Supplemental Keywords:
nerve agent, nerve gas, biodetoxification, bioremediation, insecticide., RFA, Scientific Discipline, Toxics, Water, Environmental Chemistry, pesticides, Chemistry, Bioremediation, Engineering, Environmental Engineering, agricultural runoff, organophosphorus, flavobacterium, biological treatment, biodegradation, E. Coli, chemical transport, enzyme activities, Parathion, pesticide residue, water treatmentProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.