The pyrethroid pesticides residues on foods and environmental contamination are a public safety concern. Pretreatment with pyrethroid hydrolase has the potential to alleviate the conditions. For this purpose, a fungus capable of using pyrethroid pesticides as a sole carbon source was isolated from the soil and characterized as Aspergillus niger ZD11. A novel pyrethroid hydrolase from cell extract was purified 41.5-fold to apparent homogeneity with 12.6% overall recovery. It is a monomeric structure with a molecular mass of 56 kDa, a pI of 5.4, and the enzyme activity was optimal at 45 degrees C and pH 6.5. The activities were strongly inhibited by Hg(2+), Ag(+), and rho-chloromercuribenzoate, whereas less pronounced effects (5-10% inhibition) were observed in the presence of the remaining divalent cations, the chelating agent EDTA and phenanthroline. The purified enzyme hydrolyzed various insecticides with similar carboxylester. trans-Permethrin is the preferred substrate.

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.

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