Latifi Ali Mohammad, Karami Ali, Khodi Samaneh
Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Appl Biochem Biotechnol. 2015 Oct;177(3):624-36. doi: 10.1007/s12010-015-1766-0. Epub 2015 Aug 4.
Compounds including organophosphorus pesticides (OPs) and chemical nerve agents are toxic compounds synthesized recently which disrupt the mechanisms of neural transmission. Therefore, a critical requirement is the development of a bio-refining technology to facilitate the biodegradation of organophosphorus pollutants. The diisopropylfluorophosphatase (DFPase, EC 3.1.8.2) from the ganglion and brain of Loligo vulgaris acts on P-F bonds present in some OPs. Intracellular production of OPs-degrading enzymes or the use of native bacteria and fungi leads to a low degradation rate of OPs due to a mass transfer issue which reduces the overall catalytic efficiency. To overcome this challenge, we expressed DFPase on the surface of E. coli for the first time by employing the N-terminal domain of the ice nucleation protein (InaV-N) as an anchoring motif. Tracking the recombinant protein confirmed that DFPase is successfully located on the outer membrane. Further studies on its activity to degrade diisopropylfluorophosphate (DFP) showed its significant ability for the biodegradation of diisopropylfluorophosphate (DFP) with a specific activity of 500 U/mg of wet cell weight. Recombinant cells could also degrade chlorpyrifos (Cp) with an activity equivalent to a maximum value of 381.44 U/ml with a specific activity of 476.75 U/mg of cell, analyzed using HPLC technique. The optimum activity of purified DFPase was found at 30 °C. A more increased activity was also obtained in the presence of glucose-mineral-salt (GMS) supplemented with tryptone and 100 mg/L Co(2+) ion. These results highlight the high potential of the InaV-N anchoring domain to produce an engineered bacterium that can be used in the bioremediation of pesticide-contaminated environments.
包括有机磷农药（OPs）和化学神经毒剂在内的化合物是最近合成的有毒化合物，它们会破坏神经传递机制。因此，迫切需要开发一种生物精炼技术来促进有机磷污染物的生物降解。来自普通枪乌贼神经节和大脑的二异丙基氟磷酸酶（DFPase，EC 3.1.8.2）作用于某些OPs中存在的P-F键。由于传质问题导致整体催化效率降低，细胞内产生OPs降解酶或使用天然细菌和真菌会导致OPs降解率较低。为了克服这一挑战，我们首次通过使用冰核蛋白的N端结构域（InaV-N）作为锚定基序，将DFPase表达在大肠杆菌表面。追踪重组蛋白证实DFPase成功定位在外膜上。对其降解二异丙基氟磷酸酯（DFP）的活性的进一步研究表明，它具有显著的生物降解二异丙基氟磷酸酯（DFP）的能力，比活性为500 U/mg湿细胞重量。使用HPLC技术分析，重组细胞还可以降解毒死蜱（Cp），活性相当于最大值381.44 U/ml，比活性为476.75 U/mg细胞。纯化的DFPase的最佳活性在30℃下发现。在补充了胰蛋白胨和100 mg/L Co(2+)离子的葡萄糖-矿物盐（GMS）存在下，也获得了更高的活性。这些结果突出了InaV-N锚定结构域在生产可用于农药污染环境生物修复的工程菌方面的巨大潜力。
