title: Bacterial Expression and Kinetic Analysis of Carboxylesterase 001D from Helicoverpa armigera.
abstract: Carboxylesterasesare an important class of detoxification enzymes involved in insecticide resistance in insects. A subgroup of Helicoverpa armigera esterases, known as Clade 001, was implicated in organophosphate and pyrethroid insecticide resistance due to their overabundance in resistant strains. In this work, a novel carboxylesterasegene 001D of H. armigera from China was cloned, which has an open reading frame of 1665 nucleotides encoding 554 amino acid residues. We used a series of fusion proteins to successfully express carboxylesterase 001D in Escherichia coli. Three different fusion proteins were generated and tested. The enzyme kinetic assay towards 1-naphthyl acetate showed all three purified fusion proteins are active with a Kcat between 0.35 and 2.29 s(-1), and a Km between 7.61 and 19.72 μM. The HPLC assay showed all three purified fusion proteins had low but measurable hydrolase activity towards β-cypermethrin and fenvalerate insecticides (specific activities ranging from 0.13 to 0.67 μM·min(-1)·(μM(-1)·protein)). The enzyme was stable up to 40 °C and at pH 6.0-11.0. The results imply that carboxylesterase 001D is involved in detoxification, and this moderate insecticide hydrolysis may suggest that overexpression of the gene to enhance insecticide sequestration is necessary to allow carboxylesterases to confer resistance to these insecticides in H. armigera.
authors.affiliation: Research and Development Centre of Biorational Pesticides, Northwest Agriculture and Forestry University, Yangling 712100, China. Yongqiangli@nwsuaf.edu.cn.
authors.forename: Yongqiang
authors.initials: Y
authors.lastname: Li
authors.affiliation: Commonwealth Scientific and Industrial Research Organisation Land & Water Flagship, Canberra, ACT 2601, Australia. Jian-Wei.Liu@csiro.au.
authors.forename: Jianwei
authors.initials: J
authors.lastname: Liu
authors.affiliation: Research and Development Centre of Biorational Pesticides, Northwest Agriculture and Forestry University, Yangling 712100, China. lumei_1990@nwsuaf.edu.cn.
authors.forename: Mei
authors.initials: M
authors.lastname: Lu
authors.affiliation: Research and Development Centre of Biorational Pesticides, Northwest Agriculture and Forestry University, Yangling 712100, China. zhiqingma@nwsuaf.edu.cn.
authors.forename: Zhiqing
authors.initials: Z
authors.lastname: Ma
authors.affiliation: Research and Development Centre of Biorational Pesticides, Northwest Agriculture and Forestry University, Yangling 712100, China. cchongling@nwsuaf.edu.cn.
authors.forename: Chongling
authors.initials: C
authors.lastname: Cai
authors.affiliation: Research and Development Centre of Biorational Pesticides, Northwest Agriculture and Forestry University, Yangling 712100, China. yunhongwang@hotmail.com.
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Research and Development Centre of Biorational Pesticides, Northwest Agriculture and Forestry University, Yangling 712100, China. zhxing1952@gmail.com.
authors.forename: Xing
authors.initials: X
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D043182:Carboxylesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003368:Gossypium / Q000469:parasitology; D006868:Hydrolysis; D007306:Insecticides / Q000378:metabolism; D007700:Kinetics; D007915:Lepidoptera / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011722:Pyrethrins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D007306:Insecticides; D011722:Pyrethrins; D011994:Recombinant Proteins; D043182:Carboxylesterase
keywords: Helicoverpaarmigera; carboxylesterase; heterologous expression; pyrethroid