Isolation of an enzyme from soil that degrades the organophosphorus insecticide, crotoxyphos

An enzyme which catalyzed the hydrolysis of crotoxyphos ((E)-1-phenylethyl 3-[(dimethoxyphosphinyl)oxy]-2-butenoate) was isolated from nonsterile and radiation-sterilized Chehalis clay loam with 1.5M Tris (2-(hydroxymethyl)-2-nitro-1,3-propanediol) and partially purified with lead acetate treatment. Two soil-g equivalents of lead acetate purified enzyme in pH 8 buffer hydrolyzed 0.13 mumol of substrate to dimethyl phosphate and alpha-methylbenzyl 3-hydroxycrotonate in 16 hr at 37 degrees C. The enzyme exhibited maximal activity around pH 8.0 and was irreversibly inactivated below pH 5.0 or above pH 10.0. The Km value for crotoxyphos was calculated to be 4.63 x 10(-3) M. The enzyme was stable at 60 degrees C for 10 min, retained activity indefinitely at -10 degrees C, and was completely inactivated within a week at room temperature. When applied to autoclayed Chehalis clay loam, purified enzyme lost 75% of its activity after one week and the remainder within two weeks.