Molecular Cloning, Purification, and Biochemical Characterization of a Novel Pyrethroid-Hydrolyzing Esterase from Klebsiella sp. Strain ZD112

Abstract

The gene encoding pyrethroid-hydrolyzing esterase (EstP) from Klebsiella sp. strain ZD112 was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the estP gene revealed an open reading frame of 1914 bp encoding for a protein of 637 amino acid residues. No similarities were found by a database homology search using the nucleotide and deduced amino acid sequences of the esterases and lipases. EstP was heterologously expressed in E. coli and purified. The molecular mass of the native enzyme was approximately 73 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of EstP indicated molecular masses of 73 and 73.5 kDa, respectively, suggesting that EstP is a monomer. The purified EstP not only degraded many pyrethroid pesticides and the organophosphorus insecticide malathion, but also hydrolyzed rho-nitrophenyl esters of various fatty acids, indicating that EstP is an esterase with broad substrates. The K(m) for trans- and cis-permethrin and k(cat)/K(m) values indicate that EstP hydrolyzes both these substrates with higher efficiency than the carboxylesterases from resistant insects and mammals. The catalytic activity of EstP was strongly inhibited by Hg2+, Ag+, and rho-chloromercuribenzoate, whereas a less pronounced effect (3-8% inhibition) was observed in the presence of divalent cations, the chelating agent EDTA, and phenanthroline.

Cloning of a Novel Pyrethroid-Hydrolyzing Carboxylesterase Gene from Sphingobium sp. Strain JZ-1 and Characterization of the Gene Product

“…Microorganisms play the most important role in degradation of pyrethroids in soils and sediments. Many pyrethroid-degrading microorganisms have been isolated from soils (13,16,24,27).The major routes of pyrethroid metabolism in pyrethroidresistant insects and pyrethroid-degrading microorganisms include oxidation by cytochrome P450s and ester hydrolysis by carboxylesterases (9). Carboxylesterases are a family of enzymes that are important in the hydrolysis of a large number of endogenous and xenobiotic ester-containing compounds, such as carbamates, organophosphorus pesticides, and pyrethroids.…”

“…Genes encoding the pyrethroid-hydrolyzing carboxylesterases from mouse liver microsomes and Klebsiella sp. strain ZD112 were cloned and functionally expressed (23,27).Pyrethroids differ from many other pesticides in that they contain one to three chiral centers; the chirality may arise from the acid moiety, the alcohol moiety, or both (Fig. 1).…”

SulE, a Sulfonylurea Herbicide De-Esterification Esterase from Hansschlegelia zhihuaiae S113

“…HPLC analysis was used to calculate initial rates of substrate disappearance. Hydrolytic activity for p-nitrophenyl esters was assayed according to the method described by Wu et al (44). One unit of enzyme activity was defined as the amount of enzyme that converted 1 mol of each sulfonylurea herbicide to its parent acid form.…”

“…The molecular mass of the denatured protein was determined by SDS-PAGE (25). The molecular mass of the native protein was determined by gel filtration (44). The pH range of the enzyme was determined by incubating the enzyme, with 50 M thifensulfuron-methyl as the substrate, for 30 min at a pH range of 3 to 11.…”

PbaR, an IclR Family Transcriptional Activator for the Regulation of the 3-Phenoxybenzoate 1′,2′-Dioxygenase Gene Cluster in Sphingobium wenxiniae JZ-1 T

“…Cypermethrin (Ͼ97% purity) and 3-PBA (98% purity) were purchased from J&K Scientific, Ltd. (Shanghai, China), were prepared as a 0.1 M stock solution in methanol, and were sterilized by membrane filtration (pore size, 0.22 m). The mineral salt medium (MSM) that was used in this study contained NaCl (1.0 g liter Ϫ1 ), NH 4 . Bacterial strains, oligonucleotides, plasmids, and culture conditions.…”