Microbes are rich sources of enzymes and esterases are one of the most important classes of enzymes because of their potential for application in the field of food, agriculture, pharmaceuticals and bioremediation. Due to limitations in their cultivation, only a small fraction of the complex microbial communities can be cultured from natural habitats. Thus to explore the catalytic potential of uncultured organisms, the metagenomic approach has turned out to be an effective alternative method for direct mining of enzymes of interest. Based on activity-based screening method, an esterase-positive clone was obtained from metagenomic libraries. Functional screening of a soil metagenomic fosmid library, followed by transposon mutagenesis led to the identification of a 1179 bp esterase gene, estM2, that encodes a 392 amino acids long protein (EstM2) with a translated molecular weight of 43.12 kDa. Overproduction, purification and biochemical characterization of the recombinant protein demonstrated carboxylesterase activity towards short-chain fatty acyl esters with optimal activity for p-nitrophenyl butyrate at pH 8.0 and 37 °C. Amino acid sequence analysis and subsequent phylogenetic analysis suggested that EstM2 belongs to the family VIII esterases that bear modest similarities to class C β-lactamases. EstM2 possessed the conserved S-x-x-K motif of class C β-lactamases but did not exhibit β-lactamase activity. Guided by molecular docking analysis, EstM2 was shown to hydrolyze a wide range of di- and monoesters of alkyl-, aryl- and benzyl-substituted phthalates. Thus, EstM2 displays an atypical hydrolytic potential of biotechnological significance within family VIII esterases. This study has led to the discovery of a new member of family VIII esterases. To the best of our knowledge, this is the first phthalate hydrolase (EstM2), isolated from a soil metagenomic library that belongs to a family possessing β-lactamase like catalytic triad. Based on its catalytic potential towards hydrolysis of both phthalate diesters and phthalate monoesters, this enzyme may find use to counter the growing pollution caused by phthalate-based plasticizers in diverse geological environment and in other aspects of biotechnological applications.

中文翻译：

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表征新型VIII族酯酶EstM2从土壤基因组能够水解雌激素邻苯二甲酸酯

微生物是酶的丰富来源，而酯酶是酶的最重要种类之一，因为它们在食品，农业，制药和生物修复领域的应用潜力很大。由于其种植的限制，只能从自然栖息地中培养一小部分复杂的微生物群落。因此，为了探索未培养生物的催化潜力，宏基因组学方法已成为直接开采目标酶的有效替代方法。基于活性的筛选方法，从宏基因组库中获得酯酶阳性克隆。功能性筛选土壤宏基因组化的fosmid文库，然后进行转座子诱变，从而鉴定出1179 bp的酯酶基因estM2，编码一个392个氨基酸长的蛋白质（EstM2），翻译后的分子量为43.12 kDa。重组蛋白的过量生产，纯化和生化表征证明了其对短链脂肪酰基酯的羧酸酯酶活性，在pH 8.0和37°C下对对硝基苯基丁酸酯具有最佳活性。氨基酸序列分析和随后的系统发育分析表明，EstM2属于与C类β-内酰胺酶具有适度相似性的VIII族酯酶。EstM2具有C类β-内酰胺酶的保守SxxK基序，但不具有β-内酰胺酶活性。在分子对接分析的指导下，EstM2可水解多种烷基，芳基和苄基取代邻苯二甲酸酯的二酯和单酯。从而，EstM2在VIII族酯酶中显示出非典型的具有生物技术意义的水解潜力。该研究导致发现了VIII族酯酶的新成员。据我们所知，这是第一种邻苯二甲酸酯水解酶（EstM2），从土壤宏基因组库中分离出来，该库属于一个拥有β-内酰胺酶（如催化三联体）的家族。基于其对邻苯二甲酸二酯和邻苯二甲酸单酯水解的催化潜力，该酶可用于抵抗在各种地质环境和生物技术应用其他方面由邻苯二甲酸酯类增塑剂引起的日益严重的污染。分离自土壤宏基因组文库，该数据库属于拥有β-内酰胺酶（如催化三联体）的家族。基于其对邻苯二甲酸二酯和邻苯二甲酸单酯水解的催化潜力，该酶可用于抵抗在各种地质环境和生物技术应用其他方面由邻苯二甲酸酯类增塑剂引起的日益严重的污染。从土壤宏基因组文库中分离出来，该文库属于一个拥有β-内酰胺酶（如催化三联体）的家族。基于其对邻苯二甲酸二酯和邻苯二甲酸单酯水解的催化潜力，该酶可用于抵抗在各种地质环境和生物技术应用其他方面由邻苯二甲酸酯类增塑剂引起的日益严重的污染。