Parathion hydrolase

Mulbry WW, JS Karns, PC Kearney, JO Nelson, CS McDaniel, JR Wild (1986) Identification of a plasmid-borne Parathion hydrolase gene from Flavobacterium sp. by Southern hybridization with opd from Pseudomonas diminuta. Appl Environ Microbiol 51 926-930. 

Greenhouse soil was treated at 500, 1000, 2000 and 5000 ppm with Diazinon 4E. Parathion hydrolase was added to the soil to determine the efficacy of the enzyme to rapidly degrade diazinon during a spill situation. The half-life of the diazinon in the 500 ppm treatment without enzyme present was 9.4 days while the half-life of diazinon in the 500 ppm treatment with enzyme present was one hour. The half-lives of diazinon in the 1000, 2000 and 5000 ppm treatments with enzyme present were 1.2, 5.6 and 128 hours (5.3 days), respectively. These data indicate that parathion hydrolase can be used effective " to rapidly reduce large concentrations of diazinon in soil. 

At diazinon concentrations above 2000 ppm the enzyme is less effective. Parathion hydrolase is readily soluble in water, is reasonably stable and can be easily handled in the field. Further research is needed to evaluate the efficacy of parathion hydrolase to decontaminate diazinon under actual spill conditions. 

Most of the studies done, by Munnecke were small scale laboratory studies. The efficacy of parathion hydrolase has not been tested under field conditions. It was the major objective of our study to determine the usefulness of parathion hydrolase for the decontamination of high concentrations of formulated diazinon in soil under greenhouse conditions. A secondary, but very important, objective was to determine if the enzyme could be handled in a practical fashion as would be done in the field and retain its ability to degrade diazinon. 

Materials Parathion hydrolase was obtained from Doug Munnecke (4,5). The specific activity was measured by a method of Munnecke (4,5) and was found to be 0.1 pmole diazinon hydro-lyzed/mg total protein/minute. Diazinon 4E was obtained from CIBA-GEIGY, Greensboro, NC. 

Table II. Parathion Hydrolase Studies. Enzymatic vs. Chemical Hydrolysis of Organophosphate Insecticides...

Solubility of Parathion Hydrolase During Soil Application... 

Parathion hydrolase is soluble in water and easily handled during application to soil. 

The stability of parathion hydrolase was determined for refrigerator storage and room temperature storage. The enzyme solution is stable under refrigeration ( ) The enzyme also remains stable at room temperature for long periods ( ). 

Table IV. Efficacy of Parathion Hydrolase Decontamination of Diazinon 4E Georgia Sandy Loam Soil...

Figure 5 shows the degradation of diazinon in soil at 2000 ppm by parathion hydrolase. It is easy to see that degradation is very rapid and effective - reaching safe environmental levels by 24 hours. 

One important aspect of any cleanup technique, is the type of degradation products that are produced. These products must be known in order to assess their potential environmental impact and toxicological hazards. One of the major degradation products of diazinon, oxypyrimidine was measured in soil after treatment with parathion hydrolase. Figure 6 shows that oxypyrimidine increases in soil as the diazinon is degraded by the enzyme. 

The enzyme parathion hydrolase is active enough to be used effectively to degrade high concentrations of diazinon in soil. The half-life of diazinon in soil treated at 2000 ppm was 5.6 hours. 

Oxypyrimidine is one of the degradation products after using parathion hydrolase. 

It has been proposed that parathion hydrolase, also known as organophosphorous phosphotriesterase, be used for pesticide detoxification as an alternative to more common treatment methods. Parathion hydrolase is produced by a number of bacteria including Pseudomonas sp., Flavobacte-rium sp. and a recombinant Streptomyces [17,54]. It has been shown to hydrolyze some of the most widely used organophosphate pesticides such as methyl and ethyl parathion, diazinon, fensulfothion, dursban, and couma-phos [18,19]. It should be noted that organophosphate pesticides constitute the major proportion of agricultural pesticides used at present and are implicated in an estimated 800,000 pesticide poisoning cases every year [18]. Hydrolysis accomplishes the detoxification of the pesticide and makes the products amenable to biological treatment. 

Sedar, C. M., Gibson, D. T. (1985). Enzymatic hydrolysis of organophosphates Cloning and expression of a parathion hydrolase gene from Pseudomonas diminuta. Bio/Technology, 3, 567-571. 

Arvlester Hydrolase and Parathion Hydrolase. Arylester hydrolase in mammalian serum (42.431 catalyzes very efficient hydrolysis of the organophosphorus oxons such as paraoxon (Table III). This enzyme does not hydrolyze the parent phosphorothioate insecticides such as methyl parathion, but only the oxon metabolites such as methyl paraoxon (441. This enzyme is has been referred to as phosphotriesterase however, a triester is not required as seen in the rapid hydrolysis of 10 organophosphinates by rabbit serum arylester hydrolase (44). This mechanism appears only rarely in birds... 

A parathion hydrolase has been described to hydrolyze both phosphorothioates and phosphates and its gene, opd, cloned from a plasmid carried by Psudomonas diminuta... 

Rowland SS, Speedie MK and Pogell BM (1991). Purification and characterization of a secreted recombinant phosphotriesterase (parathion hydrolase) from Streptomyces lividans. Appl Environ Microbiol, 57, 440-444. 

In addition, a leader peptide encoded by the opd gene functions to insert the processed parathion hydrolase protein in the membrane of the native Flavobacterium and Pseudomonas diminuta strains but causes the protein to be excreted by Streptomyces lividans (26,28). The ability of these regulatory elements to function across such a wide range of microorganisms is strongly suggestive of the important role that plasmid mobility and recombination have played in the dissemination of the opd gene. 

OP hydrolyzing enzymes, e.g., OPH, OPAA, paraoxonase, parathion hydrolase, etc. 

It has been demonstrated that a variety of enzymes exhibited enhanced mechanical and chemical stability when immobilized on a solid support, producing a biocatalyst. Munnecke first immobilized a pesticide detoxification extract from bacteria by absorption on glass beads. The absorbed extract retained activity for what was then a remarkable full day. Wood and co-workers, using isocyanate-based polyurethane foams (Hypol ), found that a number of enzymes unrelated to OP hydrolysis could be covalently bound to this polymer. Later, Havens and Rase immobilized a parathion hydrolase. Furthermore, Turner observed that polyurethane foams are excellent adsorption materials for OP such as pesticide vapors. ... 

Acetyl cholinesterase Polyphenol oxidase (PPO) Parathion hydrolase Tyrosinase... 

Sacks V., Eshkenazi I., Neufeld T., Dosoretz C., and Rishpon J., Immobilized parathion hydrolase An amperometric sensor for parathion. Anal. Chem., 72, 2055-2058, 2000. 

---