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

烷基酚聚氧基酸酯（APEOs）是一类非离子型表面活性剂，经细菌降解后可生成具有潜在内分泌干扰作用的短乙氧基链烷基酚。本研究为阐明APEOs的生物降解机制，从OPEO降解菌恶臭假单胞菌S-5中分离出与OPEO生物降解相关的基因，并对其DNA序列进行分析。通过将转座子Tn5插入到p.p.a putida S-5中，构建了大约11000个转座子突变体。分离出17个不能代谢OPEO的突变体，即OPEO降解基因干扰物。测定了转座子插入干扰基因的DNA序列。相似性搜索结果显示，这些基因编码的17个蛋白与酰基辅酶A合成酶、伴侣蛋白、NADH醌氧化还原酶等氨基酸序列相似性较高。RT-PCR分析了不同碳源（OPEO或葡萄糖）培养的p.p.a putida S-5细胞中9个基因的表达情况。T424基因仅在OPEO上表达，其他基因在葡萄糖和OPEO上均有表达。

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.