Diazinon hydrolase

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 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. 

The answer is D. Organophosphates react with the active site serine residue of hydrolases such as acetylcholinesterase and form a stable phosphoester modification of that serine that inactivates the enzyme toward substrate. Inhibition of acetylcholinesterase causes overstimulation of the end organs regulated by those nerves. The symptoms manifested by this patient reflect such neurologic effects resulting from the inhalation or skin absorption of the pesticide diazinon. 

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. 

Biological. Sethimathan and Yoshida (1973a) isolated a Flavobacterium sp. (ATCC 27551) from rice paddy water that metabolized diazinon as the sole caibon source. Diazinonwas readily hydrolyzed to 2-isopropyl-4-methyl-6-ltydroxypyrimidine under aerobic conditions but less rapidly under anaerobic conditions. This bacterium as well as enrichment cultures isolated from a diazinon-treated rice field mineralized the hydrolysis product to carbon dioxide (Sethunathan and Pathak, 1971 Sethunathan and Yoshida, 1973). Rosenberg and Alexander (1979) demonstrated that two strains of Pseudomonas grew on diazinon and produced diethyl phosphorothioate as the major end product. The rate of microbial degradation increased in the presence of an enzyme (parathion hydrolase), produced by a mixed culture of Pseudomonas sp. (Honeycutt et al., 1984). 

Organophosphorus hydrolase (OPH, EC 3.1.8.1) is a homodimer with a binuclear metal center. OPH has broad substrate specificity and can hydrolyze organophosphate pesticides such as methyl paradiion, ediyl parathion, paraoxon, chlorpyrifos, coumaphos, cyanophos and diazinoa Table I 9,12-14). The enzymatic hydrolysis rates are 40 - 2450 times faster than chemical hydrolysis at pH 7.0 and the enzyme is reported to be stable at ten ratures of up to 4S-S0°C (3). However, hydrolysis rates varied from very fast for phosphotriesters and phosphothiolester pesticides (P-0 bond) such as paraoxon (ken > 3800s ) and coumaphos (kcat = 800s ) to limited hydrolysis for Diazinon (kcat == 176 s ) and fensulfothion (l a, = 67 s ) (14). 

---