Isolation And Characterization Of Pyrethroid Degrading Strain JZ-2 & Purification And Characteristics Of A Pyrethroid Hydrolase

In recent years, as the replacement of more toxic and environmentally persistent organochlorine and organophosphorus pesticides, pyrethroids have been widely used for insect control on agricultural crops, and home insecticides. With such extensive use, the pollution of synthetic pyrethroids in the environment is bound to be substantial; there has been increasing concern regarding the ecological and health risk from pyrethroid residues. Due to its cost-effctiveness, convenience and security, biodegradation plays a crucial role in detoxifying pyrethroid residues in the environment. In this thesis, we will study isolation, identification, biological characteristics, degradation properties, metabolic pathway, purification of a pyrethroid hydrolase about pyrethroid-degrading strain. I hope that the preliminary conclusions will be of some assistance to eliminate the pyrethroid residues in the environment and food.By conventional enrichment culture, bacteria JZ-2 capable of degrading fenpropathrin was isolated from a pyrethroid-manufacturing wastewater bio-treatment facility. Phylogenetic analysis based on 16S rDNA gene sequences indicated that strain JZ-2 belongs to the genus Sphingobium, as they showed highest sequence similarities to Sphingobium cloacae JCM 10874T and Sphingobium ummariense CCM 7431T.Strain JZ-2 contained C18:ωo7c as the predominant fatty acid; C14:0 2-OH as the major 2-hydroxy fatty acid; Ubiquinone Q-10 as the main respiratory quinone; diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidyhnonomethylethanolamine, phosphatidylethanolamine and sphingoglycolipid as the predominant polar lipids, and spermidine as the major polyamine component. G+C mol% is 63.8±0.8 mol%. DNA-DNA hybridization results showed that strain JZ-2 had low genomic relatedness with Sphingobium cloacae JCM 10874 (34%) and Sphingobium ummariense CCM 7431T (23%), respectively. Based on the results of phenotypic (physiological & biochemical analysis, spermidine, polar lipids and ubiquinone patterns), genotypic and phylogenetic properties, strain JZ-2 represents a novel species of the genus Sphingobium, for which the name Sphingobium faniae sp. nov. is proposed. The type strain is JZ-2T(=CGMCC 1.7749T=DSM 21829T). The optimal pH for the growth of strain JZ-2 was 7.0, the optimal temperature was 30℃. The aeration was related positively to strain growth. Strain JZ-2 could tolerate 10～15 g·1-1 NaCl. Strain JZ-2 shows resistance to ampicillin and streptomycin.Strain JZ-2 was capable of degrading a wide range of pyrethroid pesticides, including fenpropathrin, permethrin, cypermethrin, fenvalerate, deltamelthrin, cyhalothrin and bifenthrin. Fenpropathrin was the most preferred substrate, permethrin and cypermethrin were degraded slower than fenpropathrin, while much faster than fenvalerate, deltamethrin, cyhalothrin and bifenthrin. Bifenthrin was the most persistent. The the substitutions of methyl with dichloroethylene or a-cyano group had no significant influence on the degradation efficiencies, while the substitutions of chloroyl with bromovinyl, fluoro, or chloridben on the chrysanthemic acid and the replacement of the 3-phenoxybenzyl with a biphenyl significantly reduced the degradation efficiencies.A novel qualitative analysis method of pyrethroid degradation was established. Compared to other methods, this method is simple, rapid, reliable and low-cost. A novel hydrolase (PytH) was purified from strain JZ-2 by ammonium sulfate precipitation, gel filtration chromatography and PAGE extraction. It was a monomeric structure with a molecular mass of 31±1 kDa, a pI of 4.8, and the enzyme activity was optimal at 40℃ and pH7.5, respectively. The enzyme was stable over a range from 10℃ to 50℃. The pyrethroid hydrolyzing activity was strongly inhibited by Ag+, Cu+, Hg2+, Ni2+, Zn+ methyl parathion and ρ-chloromercuribenzoate. The hydrolase was completely inhibited by phenylmethylsulfonyl fluoride, indicating that PytH seems a serine esterase. The most preferred substrate of PytH was fenpropathrin, specific activity was 42.31 U·mg-1, permethrin, cypermethrin, fenvalerate, deltamelthrin and bifenthrin was also hydrolyzed. The Vmax for the degradation of fenpropathrin was 7.536 nmol·min-1. And the Michaelis-Mentn’ constant (Km) was 0.05 nmol·ml-1.The amino acid sequences of the N terminus of the enzyme (NH2-Met-Thr-Val-Thr-Asp-Ile-Ile-Leu-Ile-His-Gly-Gla-Leu-Asn-Arg) were determined by automated Edman degradation. There was no homology with the previously described pyrethroid hydrolase EstP, Pye3 or with other recognized hydrolases that have been isolated from mammals and insects.The proposal metabolic pathway of pyrethroid was deduced as follows:strain JZ-2 metabolized fenpropathrin to 2,2,3,3-tetramethylcyclopropanecarboxylic acid and 3-phenoxybenzaldehyde by hydrolysis, then 3-phenoxybenzaldehyde were further oxidized to 3-phenoxy benzoate,3-phenoxy benzoate was metabolized to protocatechuate and phenol by 3-phenoxybenzoate dioxygenase; protocatechuate were degraded by protocatechuate-3,4-dioxygenase, whereas phenol was transformed to catechol and then metabolized by ortho cleavage pathway.JZ-2 was used preliminarily to study biodegradation effects on low concentration fenpropathrin in pond water, which was simulated to natural condition. When supplemented with MSM liquid medium, JZ-2 could effectively degrade 50 μg·l-1 fenpropathrin in pond water and sediment after 72h, toxicity of fenpropathrin to aquatic organisms was eliminated. The results indicated that strain JZ-2 has potential use for bioremediation of pyrethroid-contaminated water.