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

Abstract

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 μmol of substrate to dimethyl phosphate and α-methylbenzyl 3-hydroxycrotonate in 16 hr at 37°C. The enzyme exhibited maximal activity around pH 8.0 and was irreversibly inactivated below pH 5.0 or above pH 10.0. The K_m value for crotoxyphos was calculated to be 4.63×10⁻³ M. The enzyme was stable at 60°C for 10 min, retained activity indefinitely at −10°C, and was completely inactivated within a week at room temperature. When applied to autoclaved Chehalis clay loam, purified enzyme lost 75% of its activity after one week and the remainder within two weeks.