Molecular Cloning, Expression, And Characterization Of A Novel Pyrethroid-Hydrolyzing Enzyme From Sphingobium Sp. Strain JZ-1

Synthetical pyrethroids (SPs), which are synthetic version of an extract from the chrysanthemum, are now the major class of insecticides used for insect control in agriculture and households as a replacement for more toxic and environmentally persistent organochlorine and organophosphorus pesticides. Currently, SPs annual sales are estimated to reach above 1.5 billion dollars and rank the second in global insecticide sales. In China, SPs account for 1/3 of total pesticides application area.Although pyrethroid pesticides generally have lower acute oral mammalian toxicity than organophosphate and organochlorine insecticides, exposure to pyrethroid pesticides might cause damage to reproductive, immune, endocrine and cardiovascular systems. Especially, most pyrethroid pesticides possess acute toxicity to some nontarget organisms, such as bees, fish, and aquatic invertebrates, with LC50 values less than 0.5μg/L and factor of safety lower than 0.063 to fish.Great concerns have been raised about the persistence and degradation of pyrethroid pesticides in the environment. Compared with other physicochemical methods, bioremediation is a cost-effective, convenient and security solution to eliminate the pyrethroid residues in soil, sediment, and agricultural products. Although many pyrethroid-degrading microorganisms have been isolated from the environment, there were only about five pyrethrod-hydrolyzing enzymes purified and three genes of pyrethrod-hydrolyzing enzymes were cloned.A SPs-degrading bacterium, designated JZ-1, isolated from activated sludge treating SPs-manufacturing wastewater, was determined using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JZ-1T belongs to the genus Sphingobium, as it showed highest sequence similarities to Sphingobium faniae JZ-2T (98.6%), Sphingobium cloacae JCM 10874T (98.5%), Sphingobium vermicomposti VC-230T (97.4%) and Sphingobium ummariense RL-3T (96.9%). Chemotaxonomic data also supported the affiliation of the strain to the genus Sphingobium:C18:1ω7c was the predominant non-hydroxylated fatty acid and C14:0 2-OH was the major hydroxylated fatty acid; Ubiquinone Q-10 was the main respiratory quinine; diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingoglycolipids (SGL) and two unidentified phospholipids was the major polar lipids; and spermidine was the major polyamine component. Strain JZ-1 showed low DNA-DNA relatedness values with Sphingobium faniae JZ-2T (30.2%), Sphingobium cloacae JCM 10874T (23.3%), Sphingobium vermicomposti VC-230T (10.9%) and Sphingobium ummariense RL-3T(7.9%), respectively. Based on the results of phenotypic, genotypic and phylogenetic properties, strain JZ-1T represents a novel species of the genus Sphingobium, for which the name Sphingobium wenxiniae sp. nov. is proposed. The type strain is JZ-1T (=CGMCC 1.7748T=DSM 21828T).The strain JZ-1 catalyzed the hydrolysis of cypermethrin to 2,2-dimethyl-3-(2,2-dichlorovinyl)-cyclopropanecarboxylic acid and 3-phenoxybenzaldehyde, which was converted to 3-phenoxybenzoic acid. The strain JZ-1 and cell-free extract produced a visible transparent halo of pyrethroid degradation on agar plates supplemented with 150 mg/L cypermethrin. Pyrethroid hydrolase activity was detected in both cell-free extracts prepared from cells grown on LB with and without cypemethrin, indicating that pyrethroid hydrolase was expressed constitutively in strain JZ-1. The strain was capable of utilizing cypermethrin as carbon source and degrading 2-30 mg/L cypermethrin completely.A novel esterase gene, pytH, encoding a pyrethroid-hydrolyzing carboxylesterase was cloned from Sphingobium sp. JZ-1. The gene contained an open reading frame of 840 bp encoding for a protein of 280 amino acids. The G+C mol%content was 66.55%. No potential signal sequence was found. The deduced protein was compared with the known enzymes available from the Protein Data Bank (NCBI database). PytH showed the highest homology with someα/β-hydrolase fold proteins, e.g., a hydroxynitrile lyase from Hevea brasiliensis (24% identity), a carboxylesterase BioH from E. coli (23% identity), an (-) y-lactamase from Aureobacterium sp. (23% identity), and a hydrolase from Janthinobacterium sp. (20% identity). PytH contains the typical catalytic centers Ser-His-Asp/Glu and highly conserved pentapeptide GXSXG ofα/β-hydrolase fold proteins. The mutant enzymes Ser78Thr, Asp202Asn and His230Ile (catalytic centers Ser-His-Asp mutations), and Gly76Ser and Gly80Cys (highly conserved pentapeptide GXSXG mutations) did not show any specific activity against pyrethroids at all. Thus, the enzyme PytH was proposed to be a novel member ofα/β-hydrolase fold proteins.PytH was expressed in Escherichia coli BL21 (DE3) and Escherichia coli Rosseta (DE3), and was purified using Ni-nitrilotriacetic acid affinity chromatography.PytH was a monomeric structure with a molecular mass of approximately 31 kDa and a pI of 4.85. No cofactors were required for enzyme activity. The enzyme was strongly inhibited by many metal ions (Ag+, Ni2+, Cu2+, Hg2+and Zn2+; 0.5 mM), the surfactants SDS and Tween 80 (10 mM), the Ser protease inhibitor PMSF, His modifier DEPC (0.5 mM), and thiol reagent pCMB and iodoacetamide (0.5 mM); while Triton X-100 (10mM) showed only slight inhibition (20-30% inhibition), and chelating agents EDTA and 1, 10-phenanthroline (10 mM) had little effect on the enzyme activity (less than 10% inhibition).PytH was capable of hydrolyzing all of the pyrethroids tested, and hydrolysis efficiencies were dependent on the pyrethroid molecular structure, with the hydrolysis rates descending as follows:permethrin> fenpropathrin> cypermethrin> cyhalothrin> fenvalerate> deltamethrin> bifenthrin. PytH showed the highest activity with p-nitrophenyl acetate, and the activities decreased with the increase of the aliphatic chain length. No lipolytic activity was observed with esters containing an aliphatic chain length longer than six carbon atoms, indicating that PytH was an esterase and not a lipase. The specific activities for trans- and cis-permethrin (or cypermethrin) were found to be relatively similar, and stereoisomers of fenpropathrin or fenvalerate were hydrolyzed at an approximately equal rate, indicating that PytH lacked isomer selectivity for pyrethroid pesticides.