Basic Research on the Development of Detoxification Techniques of Endocrine Disrupting Chemicals, Short Ethoxy Chain-Alkylphenols

烷基酚聚乙氧基化物（APEO）属于一组非离子表面活性剂，可被细菌生物降解，然后产生短乙氧基链烷基酚，这是潜在的内分泌干扰物。在本研究中，为了阐明APEO的生物降解机制，我们通过转座子标记从OPEO降解菌恶臭假单胞菌S-5中分离出参与APEO的生物降解的基因，并分析了其DNA序列。通过将转座子 Tn5 插入恶臭假单胞菌 S-5 中，构建了大约 11,000 个转座子突变体。分离出 17 个不能代谢 OPEO 的突变体，即 OPEO 降解基因破坏体。测定了因插入转座子而破坏的基因的DNA序列。相似性检索显示，该基因编码的17个蛋白与酰基辅酶A合成酶、伴侣蛋白、NADH醌氧化还原酶等具有较高的氨基酸序列相似性。RT-PCR分析了9个基因在不同碳源（OPEO或葡萄糖）上生长的恶臭假单胞菌S-5细胞中的基因表达。 T424基因仅在OPEO上生长的细胞中表达，而其他基因在葡萄糖和OPEO上生长的细胞中表达。

Alkylphenol polyethoxylates (APEOs) belonging to a group of nonionic surfactants are biodegraded by bacteria and then short ethoxy chain-alkylphenols which are potential endocrine disrupters are produced. In this study, to elucidate the biodegradation mechanism of APEOs, we isolated genes that were involved in biodegradation of octylphenol polyethoxylate (OPEO), a kind of APEOs, from OPEO degrader Pseudomonas putida S-5 by transposon tagging and analyzed their DNA sequences. Approximately 11,000 transposon mutants were constructed by insertion of transposon Tn5 into P.putida S-5. Seventeen mutants that are not able to metabolize OPEO, namely OPEO-degradation gene disruptants, were isolated. DNA sequences of the genes disrupted by insertion of transposon were determined. A similarity search showed that 17 proteins encoded by the genes have high amino acid sequence similarity to acyl-CoA synthetase, chaperone protein, NADH quinone oxidoreductase, etc. Gene expression of 9 genes in P.putida S-5 cells grown on different carbon source (OPEO or glucose) was analyzed by RT-PCR. T424 gene was expressed only in cells grown on OPEO, while the other genes were expressed in cells grown on glucose as well as on OPEO.