Department of Biotechnology, National Kaohsiung Normal University, Kaohsiung, Taiwan.
Department of Environmental Resources Management, Chia Nan University of Pharmacy & Science, Tainan, Taiwan.
Ecotoxicol Environ Saf. 2019 May 30;173:452-460. doi: 10.1016/j.ecoenv.2019.02.056. Epub 2019 Feb 21.
Although laccase is involved in the biotransformation of 2,4,6-trinitrotoluene (TNT), little is known regarding the effect of E. coli laccase on TNT biotransformation. In this study, E. coli K12 served as the parental strain to construct a laccase deletion strain and two laccase-overexpressing strains. These E. coli strains were used to investigate the effect of laccase together with copper ions on the efficiency of TNT biotransformation, the variety of TNT biotransformation products generated and the toxicity of the TNT metabolites. The results showed that the laccase level was not relevant to TNT biotransformation in the soluble fraction of the culture medium. Conversely, TNT metabolites varied in the insoluble fraction analyzed by thin-layer chromatography (TLC). The insoluble fraction from the laccase-null strain showed fewer and relatively fainter spots than those detected in the wild-type and laccase-overexpressing strains, indicating that laccase expression levels were interrelated determinants of the varieties and amounts of TNT metabolites produced. In addition, the aquatic invertebrate Tigriopus japonicus was used to assess the toxicity of the TNT metabolites. The toxicity of the TNT metabolite mixture increased when the intracellular laccase level in strains increased or when purified E. coli recombinant Laccase (rLaccase) was added to the culture medium. Thus, our results suggest that laccase activity must be considered when performing microbial TNT remediation.
尽管漆酶参与了 2,4,6-三硝基甲苯（TNT）的生物转化，但对于大肠杆菌漆酶对 TNT 生物转化的影响知之甚少。在这项研究中，大肠杆菌 K12 被用作构建漆酶缺失菌株和两种漆酶过表达菌株的亲本菌株。这些大肠杆菌菌株被用于研究漆酶与铜离子一起对 TNT 生物转化效率、生成的 TNT 生物转化产物的种类和 TNT 代谢物的毒性的影响。结果表明，漆酶水平与培养基中可溶性部分的 TNT 生物转化无关。相反，通过薄层层析（TLC）分析的不溶性部分中 TNT 代谢物的变化。与野生型和漆酶过表达菌株相比，漆酶缺失菌株的不溶性部分显示出较少且相对较淡的斑点，这表明漆酶表达水平是产生 TNT 代谢物的种类和数量的相互关联的决定因素。此外，还使用水生无脊椎动物日本虎斑猛水蚤来评估 TNT 代谢物的毒性。当菌株中细胞内漆酶水平增加或向培养基中添加纯化的大肠杆菌重组漆酶（rLaccase）时，TNT 代谢物混合物的毒性增加。因此，我们的结果表明，在进行微生物 TNT 修复时必须考虑漆酶活性。
