Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst).
Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst).
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DOI：
10.1371/journal.pone.0158146
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Araujo AP
中科院分区：
文献类型：
--
作者：
Lopes JL;Yoneda JS;Martins JM;DeMarco R;Jameson DM;Castro AM;Bossolan NR;Wallace BA;Araujo AP
从油藏中发现的嗜热生物中分离出的酶可以在许多领域得到应用，包括石油化工、制药、生物能源和食品/乳制品工业。本研究对极端细菌运动石油杆菌的一种酯酶(命名为PmEst)进行了电子鉴定和重组生产。然后结合同步辐射圆二色谱(SRCD)和荧光光谱对PmEst在不同底物上的稳定性和水解性进行了生化、生物信息学和结构表征。该酶对对硝基丁酸苯酯具有较高的米氏常数(Km0.16 mM)和~55℃的最适酶活。PmEst的二级结构在酸性条件下保持不变，而在碱性条件下则不保存。PmEst在高浓度尿素或胍的作用下通过明显不同的机制被解离。在乙醇或丙醇存在下，PmEst的酯酶活性保持不变，而在这些有机溶剂存在下，PmEst的熔融温度提高约8℃。PmEst是一种中温酯酶，底物偏爱中短链。PmEst的SRCD数据与α/β蛋白的预测一致，这导致我们假设它显示了该家族的典型酯酶折叠。酶在有机溶剂中稳定性的提高可能使其在合成生物学中的新应用成为可能。综上所述，我们的结果显示了PmEst酶的特点，表明它可能适合于工业过程中的应用，特别是当需要使用极性有机溶剂时。
Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required.