Microbial Cell Factories Biomed Central

Microbial Cell Factories BioMed Central Research Open Access Molecular cloning and characterization of a novel pyrethroid-hydrolyzing esterase originating from the Metagenome Gang Li†, Kui Wang† and Yu Huan Liu* Address: State Key Laboratory of Biocontrol, School of Life sciences, Sun Yat-sen University, Guangzhou, 510275, PR China Email: Gang Li - [email protected]; Kui Wang - [email protected]; Yu Huan Liu* - [email protected] * Corresponding author †Equal contributors Published: 30 December 2008 Received: 27 October 2008 Accepted: 30 December 2008 Microbial Cell Factories 2008, 7:38 doi:10.1186/1475-2859-7-38 This article is available from: http://www.microbialcellfactories.com/content/7/1/38 © 2008 Li et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Pyrethroids and pyrethrins are widely used insecticides. Extensive applications not only result in pest resistance to these insecticides, but also may lead to environmental issues and human exposure. Numerous studies have shown that very high exposure to pyrethroids might cause potential problems to man and aquatic organisms. Therefore, it is important to develop a rapid and efficient disposal process to eliminate or minimize contamination of surface water, groundwater and agricultural products by pyrethroid insecticides. Bioremediation is considered to be a reliable and cost-effective technique for pesticides abatement and a major factor determining the fate of pyrethroid pesticides in the environment, and suitable esterase is expected to be useful for potential application for detoxification of pyrethroid residues. Soil is a complex environment considered as one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms in nature are inaccessible as they are uncultivable in the laboratory. Metagenomic approaches provide a powerful tool for accessing novel valuable genetic resources (novel enzymes) and developing various biotechnological applications. Results: The pyrethroid pesticides residues on foods and the environmental contamination are a public safety concern. Pretreatment with pyrethroid-hydrolyzing esterase has the potential to alleviate the conditions. To this end, a pyrethroid- hydrolyzing esterase gene was successfully cloned using metagenomic DNA combined with activity-based functional screening from soil, sequence analysis of the DNA responsible for the pye3 gene revealed an open reading frame of 819 bp encoding for a protein of 272 amino acid residues. Extensive multiple sequence alignments of the deduced amino acid of Pye3 with the most homologous carboxylesterases revealed moderate identity (45–49%). The recombinant Pye3 was heterologously expressed in E. coli BL21(DE3), purified and characterized. The molecular mass of the native enzyme was approximately 31 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of the Pye3 indicated molecular mass of 31 kDa and 31.5 kDa, respectively, suggesting that the Pye3 is a monomer. The purified Pye3 not only degraded all pyrethroid pesticides tested, but also hydrolyzed ρ-nitrophenyl esters of medium-short chain fatty acids, indicating that the Pye3 is an esterase with broader specificity. The Km values for trans-Permethrin and cis-permethrin are 0.10 μM and 0.18 μM, respectively, and these catalytic properties were superior to carboxylesterases from resistant insects and mammals. The catalytic activity of the Pye3 was strongly inhibited by Hg2+, Ag+, ρ-chloromercuribenzoate, whereas less pronounced effect was observed in the presence of divalent cations, the chelating agent EDTA and phenanthroline. Conclusion: A novel pyrethroid-hydrolyzing esterase gene was successfully cloned using metagenomic DNA combined with activity-based functional screening from soil, the broader substrate specificities and higher activity of the pyrethroid-hydrolyzing esterase (Pye3) make it an ideal candidate for in situ for detoxification of pyrethroids where they cause environmental contamination problems. Consequently, metagenomic DNA clone library offers possibilities to discover novel bio-molecules through the expression of genes from uncultivated bacteria. Page 1 of 10 (page number not for citation purposes) Microbial Cell Factories 2008, 7:38 http://www.microbialcellfactories.com/content/7/1/38 Background sis of carboxylester linkage, and demonstrated that these Pyrethroids and pyrethrins are widely used insecticides. enzymes differ in substrate specificity, sensitivity to inhib- Natural pyrethrins are compounds with insecticidal activ- itors, activation by metals and molecular mass. In a word, ity extracted from chrysanthemum flowers. Pyrethroids this method only focuses on the culturable portion of are synthetic compounds similar in structure to microorganisms. In fact, most microorganisms have not pyrethrins, which have been modified over the years to been researched due to limitations in culturing microor- enhance their insecticidal activity and/or their persistence ganisms, it is estimated that more than 99% of microor- in the environment [1] ganisms are thought to be unculturable or difficult to culture in any given environment using standard cultiva- Pyrethroids are likely to become more widely used as tion methods and therefore not accessible as a source for organophosphate insecticides including diazinon and finding useful biomolecules [12,13]. In order to search for chlorpyrifos are phased out due to the concerns regarding new or improved bioactive products, the development of their safety [2]. Extensive applications not only result in metagenomic technologies over the past few years has pest resistance to these insecticides, but also may lead to provided access to much of the prokaryotic genetic infor- environmental issues and human exposure. A variety of mation available in environmental samples, independent personnel are exposed to pyrethroids during manufacture of culturability [14]. Through metagenomic cloning, and application, diet, and drinking water. Although these some genes encoding metagenomic lipases and esterases compounds are widely considered safe for humans [3], have been identified in metagenomic libraries from differ- numerous studies have shown that very high exposure to ent environmental samples such as soil, water, thermal pyrethroids might cause potential problems to man [4]. environment and deep-sea hypersaline anoxic basin [15- Such effects include suppressive effects on the immune 18]. system, endocrine disruption, lymph node and splenic damage, and carcinogenesis [5]. In addition, most syn- In this report, we constructed a metagenomic library from thetic pyrethroids possess apparent toxicity to fish and vegetable soil for the isolation by functional expression other aquatic organisms, including aquatic invertebrates, screening of plasmid clones with esterase activity. Several often at concentrations less than 1 μg L-1 [6]. Therefore, it clones with esterase activity were detected, and a novel is important to develop a rapid and efficient disposal pyrethroid-hydrolyzing esterase with higher activity and process to eliminate or minimize contamination of sur- broader substrate specificities was selected for further face water, groundwater and agricultural products by pyre- characterization including thermal stability, optimal tem- throid insecticides. perature and substrate specificity. To our knowledge, this is the first report so far on information about pyrethroid- Bioremediation strategies have potential for eliminating hydrolyzing esterase gene from the unculturable bacterial or reducing these residues. One such strategy would use genome. Further study is helpful to obtain excellent enzymes to degrade the pesticide residues, for example, in detoxifying enzyme for use as bioremediation agents. bioreactors through which contaminated water could be passed, or in washing solutions after post-harvest disinfes- Results tation of fruit, vegetables or animal products to reduce res- Construction of a metagenomic library and screening of a idue levels and withholding times [1]. Suitable enzymes novel esterase gene for degrading pesticide residues include hydrolases from The prokaryotic DNA for metagenomic library, which was bacteria, vertebrates and insecticide resistant insects [7-9]. used in all experiments, was extracted from vegetable soil. A novel soluble esterase of Pye3 on an activity based strat- Carboxylesterases represent a group of highly variable and egy was designed. To test the quality of the library, 100 multifunctional hydrolytic enzymes. They have potential clones were randomly selected, and the recombinant plas- for use in the hydrolysis and synthesis of important ester mids were prepared. The average insert size was 4.2 kb, compounds of pharmaceutical, food, biochemical, and and sizes ranged from 3 to 8 kb. Out of approximately biological interests. Some pyrethroid carboxylesterases 93,000 colonies, 6 clones were identified by their bright from pyrethroid-resistant insects have been purified and blue color. The metagenomic library represented about characterized [8]. Some pyrethroid-degrading bacteria 390 Mb of soil microbial community DNA. From these including Bacillus

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