Department of Life Sciences, National Central University, Jhongli, Taiwan.
Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan.
Sci Rep. 2019 May 2;9(1):6827. doi: 10.1038/s41598-019-43266-8.
Bacteria in the environment play a major role in the degradation of widely used man-made recalcitrant organic compounds. Pseudomonas nitroreducens TX1 is of special interest because of its high efficiency to remove nonionic ethoxylated surfactants. In this study, a novel approach was demonstrated by a bacterial enzyme involved in the formation of radicals to attack ethoxylated surfactants. The dihydrolipoamide dehydrogenase was purified from the crude extract of strain TX1 by using octylphenol polyethoxylate (OPEO) as substrate. The extent of removal of OPEOs during the degradation process was conducted by purified recombinant enzyme from E. coli BL21 (DE3) in the presence of the excess of metal mixtures (Mn, Mg, Zn, and Cu). The metabolites and the degradation rates were analyzed and determined by liquid chromatography-mass spectrometry. The enzyme was demonstrated to form Fenton reagent in the presence of an excess of metals. Under this in vitro condition, it was shown to be able to shorten the ethoxylate chains of OPEO. After 2 hours of reaction, the products obtained from the degradation experiment revealed a prominent ion peak at m/z = 493.3, namely the ethoxylate chain unit is 6 (OPEO) compared to OPEO (m/z = 625.3), the main undegraded surfactant in the no enzyme control. It revealed that the concentration of OPEO and OPEO decreased by 90% and 40% after 4 hours, respectively. The disappearance rates for the OPEO homologs correlated to the length of the exothylate chains, suggesting it is not a specific enzymatic reaction which cleaves one unit by unit from the end of the ethoxylate chain. The results indicate the diverse and novel strategy by bacteria to catabolize organic compounds by using existing housekeeping enzyme(s).
环境中的细菌在降解广泛使用的人工难降解有机化合物方面发挥着重要作用。假单胞菌 nitroreducens TX1 因其高效去除非离子型乙氧基化表面活性剂而备受关注。在本研究中，展示了一种新方法，该方法涉及参与形成自由基以攻击乙氧基化表面活性剂的细菌酶。二氢硫辛酰胺脱氢酶从 TX1 菌株的粗提物中通过使用辛基酚聚氧乙烯醚（OPEO）作为底物进行纯化。在过量金属混合物（Mn、Mg、Zn 和 Cu）存在下，从大肠杆菌 BL21（DE3）中纯化的重组酶对 OPEOs 在降解过程中的去除程度进行了研究。通过液相色谱-质谱分析和确定了代谢物和降解速率。该酶在过量金属存在下被证明能形成芬顿试剂。在这种体外条件下，它被证明能够缩短 OPEO 的乙氧基链。反应 2 小时后，降解实验得到的产物在 m/z=493.3 处显示出一个明显的离子峰，即乙氧基链单元为 6（OPEO），而 OPEO（m/z=625.3）是无酶对照中主要未降解的表面活性剂。这表明 OPEO 和 OPEO 的浓度分别在 4 小时后降低了 90%和 40%。OPEO 同系物的消失速率与乙氧基链的长度相关，表明这不是一种从乙氧基链末端逐个单元切割的特异性酶反应。结果表明，细菌通过利用现有的管家酶（s）对有机化合物进行代谢的多样性和新颖策略。
