title: Purification, characterization, and sensitivity to pesticides of carboxylesterase from Dendrolimus superans (Lepidoptera: Lasiocampidae).
abstract: Through a combination of steps including centrifugation, ammonium sulfate gradient precipitation, sephadex G-25 gel chromatography, diethylaminoethyl cellulose 52 ion-exchange chromatography and hydroxyapatite affinity chromatography, carboxylesterase (CarE, EC3.1.1.1) from sixth instar larch caterpillar moth, Dendrolimus superans (Lepidoptera: Lasiocampidae) larvae was purified and its biochemical properties were compared between crude homogenate and purified CarE. The final purified CarE after hydroxyapatite chromatography had a specific activity of 52.019 μmol/(min·mg protein), 138.348-fold of crude homogenate, and the yield of 2.782%. The molecular weight of the purified CarE was approximately 84.78 kDa by SDS-PAGE. Three pesticides (dichlorvos, lambda-cyhalothrin, and avermectins) showed different inhibition to crude CarE and purified CarE, respectively. In vitro median inhibitory concentration indicated that the sensitivity of CarE (both crude homogenate and final purified CarE) to pesticides was in decreasing order of dichlorvos > avermectins > lambda-cyhalothrin. By the kinetic analysis, the substrates alpha-naphthyl acetate (α-NA) and beta-naphthyl acetate (β-NA) showed lesser affinity to crude extract than purified CarE. The results also indicated that both crude homogenate and purified CarE had more affinity to α-NA than to β-NA, and the Kcat and Vmax values of crude extract were lower than purified CarE using α-NA or β-NA as substrate.
authors.affiliation: School of Forestry, Northeast Forestry University, Harbin 150040, P.R. China.
authors.forename: Chuan-shan
authors.initials: CS
authors.lastname: Zou
authors.affiliation: School of Forestry, Northeast Forestry University, Harbin 150040, P.R. China.
authors.forename: Chuan-wang
authors.initials: CW
authors.lastname: Cao
authors.affiliation: School of Forestry, Northeast Forestry University, Harbin 150040, P.R. China.
authors.forename: Guo-cai
authors.initials: GC
authors.lastname: Zhang
authors.affiliation: School of Forestry, Northeast Forestry University, Harbin 150040, P.R. China zhiyingwangnefu@aliyun.com.
authors.forename: Zhi-ying
authors.initials: ZY
authors.lastname: Wang
mesh_terms: D000818:Animals; D043182:Carboxylesterase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism*; D004006:Dichlorvos / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007306:Insecticides / Q000494:pharmacology*; D007559:Ivermectin / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D007814:Larva / Q000201:enzymology; D008970:Molecular Weight; D009036:Moths / Q000201:enzymology*; D009570:Nitriles / Q000494:pharmacology; D010575:Pesticides; D011722:Pyrethrins / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D007306:Insecticides; D009570:Nitriles; D010575:Pesticides; D011722:Pyrethrins; D007559:Ivermectin; C019264:avermectin; D004006:Dichlorvos; D043182:Carboxylesterase; C037304:cyhalothrin
keywords: Dendrolimus superans; biochemical characteristic; carboxylesterase; pesticide sensitivity; purification