Characterization of EST3: a metagenome-derived esterase with suitable properties for biotechnological applications
Characterization of EST3: a metagenome-derived esterase with suitable properties for biotechnological applications
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DOI：
10.1007/s00253-016-7385-z
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发表时间：
2016-02
影响因子：
5
通讯作者：
Thaís Carvalho Maester;M. Pereira;E. G. Machado Sierra;A. Balan;E. G. Lemos
中科院分区：
文献类型：
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作者：
Thaís Carvalho Maester;M. Pereira;E. G. Machado Sierra;A. Balan;E. G. Lemos
来自不同环境的宏基因组文库是许多脂肪酶和酯酶的广泛来源；然而，大多数这些酶在生化方面仍未得到表征。我们之前从专门用于柴油降解的微生物联盟构建了宏基因组 fosmid 文库，并测试了它的脂肪分解活性。在本研究中，我们鉴定了经过亚克隆和测序的 PL14.H10 克隆，从而能够鉴定 EST3 蛋白。该酶与来自 Parvibaculum lavamentivorans 的未表征的 α/β 水解酶表现出 74% 的氨基酸一致性 [GenBank: WP012110575.1]，并被归类为脂肪分解酶家族 IV。生化表征表明，EST3 在较宽的温度范围内表现出高活性，其中 41 至 45 °C 的活性最高。此外，这种热稳定性酯酶在弱酸性到碱性条件下均可发挥作用，最适 pH 值为 6.0。该酶对不同链长的对硝基苯酯均表现出活性，对对硝基苯辛酸酯的催化效率最高。在 0.5 mM Mn+2、Li+、EDTA 和 1% CTAB 存在下，该蛋白质的活性增加，并且在 10% 甲醇和乙醇存在下，其活性减半。此外，建立了 EST3 的同源模型并与其他酯酶进行比较，揭示了适合多种底物的底物通道。总而言之，这项工作中提供的数据揭示了 EST3 独特且有趣的特征，可以探索这些特征以在生物技术应用中进一步使用。
Metagenomic libraries from diverse environments have been extensive sources of many lipases and esterases; nevertheless, most of these enzymes remain biochemically uncharacterized. We previously built a metagenomic fosmid library from a microbial consortium specialized for diesel oil degradation and tested it for lipolytic activity. In the present study, we identified the PL14.H10 clone that was subcloned and sequenced, which enabled the identification of the EST3 protein. This enzyme exhibited 74 % amino acid identity with the uncharacterized alpha/beta hydrolase fromParvibaculum lavamentivorans[GenBank: WP012110575.1] and was classified into lipolytic enzyme family IV. Biochemical characterization revealed that EST3 presents high activity in a wide range of temperature with highest activity from 41 to 45 °C. Also, this thermostable esterase acts from mild acidic to alkaline conditions with an optimum pH of 6.0. The enzyme exhibited activity againstp-nitrophenyl esters of different chain lengths and highest catalytic efficiency againstp-nitrophenyl caprylate. The activity of the protein was increased in the presence of 0.5 mM of Mn+2, Li+, EDTA, and 1 % of CTAB and exhibited half of the activity in the presence of 10 % methanol and ethanol. Moreover, the homology model of EST3 was built and compared to other esterases, revealing a substrate channel that should fit a wide range of substrates. Taken together, the data presented in this work reveal the unique and interesting characteristics of EST3 that might be explored for further use in biotechnological applications.