Purification and characterization of carbon-phosphorus bond-cleavage enzyme from glyphosate degrading Pseudomonas putida T5.

An inducible, carbon-phosphorus bond-cleavage enzyme was purified from cells of Pseudomonas putida T5 grown on N-phosphonomethyl glycine. The native enzyme had a molecular mass of approximately 70 kD and upon sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), yielded a homogeneous protein band with an apparent molecular mass of about 70 kD. Activity of purified enzyme was increased by 627-fold compared to the crude extract and showed pH and temperature optima of approximately 7 and 30°C, respectively. The purified enzyme had an apparent Km and Vmax of 3.7 mM and 6.8 mM/min, respectively, for its sole substrate N-phosphonomethyl glycine. The enzyme was inhibited by phenylmethylsulfonyl fluoride (PMSF), indicating the presence of serine at the active site. The enzyme was not inhibited by SDS, suggesting the absence of disulfide linkage in the enzyme. The enzyme was found to be inhibited by most of the metals studied except Mg(2+). Detergents studied also inhibited glyphosate acting as a carbon-phosphorus bond-cleavage enzyme. Thus initial characterization of the purified enzyme suggested that it could be used as a potential candidate for glyphosate bioremediation.

Detection of isoprothiolane in food, soil, and water samples by immunosorbent assay using avian antibodies.

A simple competitive immunoassay was developed for the measurement of isoprothiolane in rice, soil, and water samples. It employed the avian antibodies (IgY) that recognized isoprothiolane as a capture reagent and isoprothiolane-alkaline phosphatase conjugate as an enzyme label. The assay depended on the competitive binding between the anti-isoprothiolane antibody and isoprothiolane derived from rice, soil, and water samples for binding sites with immobilized isoprothiolane-ovalbumin (OVA) conjugate. The concentration of isoprothiolane in the rice, soil, and water samples was quantified by the ability of the pesticide present in the samples to inhibit the binding of the enzyme conjugate to the antibody and subsequently the color formation in the assay. The assay was specific to isoprothiolane with a limit of detection of 2 ng/mL. Mean analytical recovery of isoprothiolane in different rice matrices was 87.20%-98.02%, for soil samples recovery was 74.24%-111.20%, and water samples recovery was 35.2%-95.73%. The precision of the assay was satisfactory. The assay compared favorably with gas chromatography (GC) in its ability to accurately measure isoprothiolane in the different rice, soil, and water samples.

Degradation of isoprothiolane by a defined microbial consortium using response surface methodology.

A defined microbial consortium was developed for the degradation of isoprothiolane. Isoprothiolane-biodegradation parameters were optimized using Response Surface Methodology (RSM). Three variables chosen for the study were inoculum concentration (50-1500 microg protein ml(-1)), temperature (25-35 degrees C) and pH (4-8) each at levels -1.682, -1, 0, 1 and 1.682. Incubation time of 72 hr was kept constant. Degradation of different concentrations of isoprothiolane was studied. The optimized conditions obtained were, inoculum concentration of 50 microg protein ml(-1) at 30 degrees C and pH between 4-8. The maximum predicted percentage degradation of 100, 100, 100, 100 and 95.5 was obtained respectively for 5, 10, 20, 30 and 50 ppm of initial isoprothiolane concentrations at different pH levels 7.7, 6.8, 6.2, 4.7 and 4.6. Validation of the model indicated that experimental values were found to be in agreement with the predicted one.