Characterization of a novel family VIII esterase EstM2 from soil metagenome capable of hydrolyzing estrogenic phthalates
Characterization of a novel family VIII esterase EstM2 from soil metagenome capable of hydrolyzing estrogenic phthalates
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DOI：
10.1186/s12934-020-01336-x
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发表时间：
2020-03-24
影响因子：
6.4
通讯作者：
Dutta, Tapan K.
中科院分区：
文献类型：
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作者：
Sarkar, Jayita;Dutta, Arindam;Dutta, Tapan K.
微生物是酶的丰富来源，酯酶是最重要的一类酶，因为它们在食品、农业、制药和生物修复领域中具有应用潜力。由于其培养的限制，只有一小部分复杂的微生物群落可以从自然栖息地培养。因此，为了探索未培养生物体的催化潜力，宏基因组学方法已被证明是直接挖掘感兴趣的酶的有效替代方法。基于活性筛选方法，从宏基因组文库中获得酯酶阳性克隆。结果通过对土壤宏基因组fosmid文库的功能筛选和转座子诱变，获得了一个1179 bp的酯酶基因estM 2，编码392个氨基酸，翻译分子量为43.12 kDa。重组蛋白的过量生产、纯化和生化表征表明，在pH 8.0和37 ℃下，对短链脂肪酰基酯具有羧酸酯酶活性，对丁酸对硝基苯基酯具有最佳活性。氨基酸序列分析和随后的系统发育分析表明，EstM 2属于VIII家族酯酶，与C类β-内酰胺酶具有适度的相似性。EstM 2具有C类β-内酰胺酶的保守S-x-x-K基序，但不显示β-内酰胺酶活性。分子对接分析的指导下，EstM 2被证明可以水解广泛的烷基，芳基和苄基取代的邻苯二甲酸酯的二酯和单酯。因此，EstM 2显示出在家族VIII酯酶中具有生物技术意义的非典型水解潜力。结论这项研究发现了VIII酯酶家族的一个新成员。据我们所知，这是第一个邻苯二甲酸酯水解酶（EstM 2），分离自土壤宏基因组文库，属于一个家庭拥有β-内酰胺酶样催化三联体。基于其对邻苯二甲酸酯二酯和邻苯二甲酸酯单酯的水解的催化潜力，该酶可用于对抗由邻苯二甲酸酯基增塑剂在不同地质环境和生物技术应用的其他方面中引起的日益严重的污染。
Background 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. Results 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 degrees 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 beta-lactamases. EstM2 possessed the conserved S-x-x-K motif of class C beta-lactamases but did not exhibit beta-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. Conclusions 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 beta-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.