Molecular cloning and characterization of a novel pyrethroid-hydrolyzing esterase originating from the Metagenome.
Molecular cloning and characterization of a novel pyrethroid-hydrolyzing esterase originating from the Metagenome.
复制标题
源自元基因组的新型拟除虫菊酯 - 氢化酯酶的分子克隆和表征。
DOI：
10.1186/1475-2859-7-38
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发表时间：
2008-12-30
影响因子：
6.4
通讯作者：
Liu YH
中科院分区：
文献类型：
--
作者：
Li G;Wang K;Liu YH
拟除虫菊酯和拟除虫菊酯是广泛使用的杀虫剂。广泛使用不仅导致害虫对这些杀虫剂产生抗性，而且可能导致环境问题和人类暴露。大量研究表明，大量接触拟除虫菊酯可能会对人类和水生生物造成潜在的问题。因此，必须发展一种快速和有效的处理程序，以消除或尽量减少拟除虫菊酯类杀虫剂对地表水、地下水和农产品的污染。生物修复被认为是一种可靠、经济的农药减排技术，也是决定拟除虫菊酯类农药在环境中命运的主要因素，合适的酯酶有望在拟除虫菊酯类农药残留脱毒方面有潜在的应用前景。土壤是一个复杂的环境，被认为是地球上微生物多样性的主要储存库之一。然而，自然界中的大多数微生物是不可接近的，因为它们无法在实验室中培养。宏基因组方法为获取新的有价值的遗传资源（新酶）和开发各种生物技术应用提供了有力的工具。拟除虫菊酯类农药在食品中的残留和环境污染是一个令人关注的公共安全问题。用拟除虫菊酯水解酯酶预处理有可能缓解这些症状。为此，利用元基因组DNA结合基于活性的功能筛选，从土壤中成功克隆出拟除虫菊酯水解酯酶基因，对pye3基因的DNA进行序列分析，发现一个819 bp的开放阅读框，编码272个氨基酸残基的蛋白。对推断的Pye3氨基酸与大多数同源羧酸酯酶进行了广泛的多序列比对，结果显示其同源性中等（45-49%）。重组蛋白Pye3在大肠杆菌BL21（DE3）中异种表达，并进行了纯化和鉴定。经凝胶过滤测定，天然酶的分子量约为31 kDa。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和氨基酸序列分析，Pye3的分子量分别为31 kDa和31.5 kDa，为单体。纯化后的Pye3不仅能降解所有拟除虫菊酯类农药，还能水解中短链脂肪酸的ρ-硝基苯基酯，表明Pye3是一种特异性较广的酯酶。反式氯菊酯和顺式氯菊酯的Km值分别为0.10 μM和顺式氯菊酯的Km值分别为0.18 μM，其催化性能优于抗性昆虫和哺乳动物羧酸酯酶。Hg2+、Ag+、ρ-氯脲苯甲酸盐对Pye3的催化活性有较强的抑制作用，而二价阳离子、螯合剂EDTA和邻菲罗啉对Pye3的催化活性影响较小。利用元基因组DNA结合基于活性的功能筛选技术，从土壤中成功克隆出一种新的拟除虫菊酯水解酯酶基因，该基因具有更广泛的底物特异性和更高的活性，使其成为原位解毒拟除虫菊酯的理想候选基因。因此，宏基因组DNA克隆文库提供了通过从未培养的细菌中表达基因来发现新的生物分子的可能性。
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. 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. 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.
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影响因子：
1.6
作者：
Alvarez-Macarie, E;Augier-Magro, V;Baratti, J
通讯作者：
Baratti, J
影响因子：
6.1
作者：
Park, EK;Kim, JH;Hammock, BD
通讯作者：
Hammock, BD
DOI：
10.1016/j.bbrc.2008.03.102
发表时间：
2008-05-30
影响因子：
3.1
作者：
Lee, Chang-Muk;Yeo, Yun-Soo;Yoon, Sang-Hong
通讯作者：
Yoon, Sang-Hong
影响因子：
4.4
作者：
Lee, Mi-Hwa;Lee, Choong-Hwan;Yoon, Jung-Hoon
通讯作者：
Yoon, Jung-Hoon
影响因子：
4.7
作者：
Chiang, SW;Sun, CN
通讯作者：
Sun, CN