PURIFICATION AND CHARACTERIZATION OF CARBOXYLAMIDASE FROM THE FALL ARMYWORM, SPODOPTERA-FRUGIPERDA (SMITH,J.E.)

Carboxylamidase which hydrolyzes p-nitroacetanilide was purified 256-f old from larval midguts of the fall armyworm, Spodoptera frugiperda (J . E. Smith), by ammonium sulfate fractionation, gel filtration, ion ex change chromatography, and hydroxyapatite chromatography, The purified enzyme was a monomer with a molecular weight of 59,000-60,000. The en zyme had an apparent K-m value of 0.63 mM and a V-max of 526.3 nmol/mi n/mg protein. It was inhibited by the hydrolase inhibitors paraoxon, t riphenyl phosphate, eserine, and phenylmethylsulfonyl fluoride, showin g 150 values of 4.7 mu M, 0.2 mM, 16 mu M, and 90 mu M, respectively. The enzyme was also completely inhibited by the organophosphorus insec ticides profenofos and dichlorvos ar 0.1 mM. The enzyme was active tow ard other amides, such as acetanilide and phenacetin, and various alph a- and beta-naphtholic esters. Based on the purification factor, subst rate specificity and sensitivity to hydrolase inhibitors, the carboxyl amidase appeared to be different from carboxylesterases in this insect . (C) 1998 Academic Press.