辛基苯酚聚氧乙基醇 (octylphenol polyethoxylates，OPEOn)為一常用於農業、工業以及一般家庭活動之難分解的非離子性界面活性劑。一旦排放至自然環境時，經常形成辛基苯酸，在結構上類似雌激素，具環境荷爾蒙效力。然而目前對於異化辛基苯酚聚氧乙基醇之酵素的瞭解仍非常有限，本實驗室已分離出可利用辛基苯酚聚氧乙基醇為唯一生長碳源之菌株Pseudomonas nitroreducens TX1，並利用一維及二維膠體電泳分析菌株P. nitroreducens TX1生長於以OPEOn為唯一碳源時所增加或減少表現的蛋白質，為了進一步證明上述發現之增加或減少表現蛋白質的合理性，本研究續以基因體學為研究方法參考全基因體已定序之Pseudomonas sp.，設計引子選殖參與OPEOn代謝及抗逆境反應的相關基因，並以反轉錄聚合酶鏈反應(RT-PCR)觀察其mRNA的表現量是否與一維及二維膠體電泳分析上所發現的蛋白質表現情形相符。本研究計成功地選殖出一個完整序列的基因及七個部份序列的基因，分別包括Arginine / Ornithine transport system substrate-binding proetin、periplasmic binding protein of ATP binding cassette transporter及TolB protein等三個運輸蛋白基因，dihydrolipoamide dehydrogenase所具有之三個與TCA cycle相關的基因群組，一個抗逆境蛋白質superoxide dismutase的基因及一個脂肪酸合成酵素Acetyl-CoA carboxylase carboxyl transferase的基因，另外，反轉錄聚合酶鏈反應的結果顯示，除了Arginine / Ornithine transport system substrate-binding proetin及periplasmic binding protein of ATP binding cassette transporter二個運輸蛋白外，其他蛋白質的基因表現皆與先前在一維及二維膠體電泳上所觀察到的蛋白質表現情形一致，其中，與高分子化合物輸送有關的TolB，其基因在mRNA表現量為選殖運輸蛋白中唯一有表現量增加的蛋白質，至於dihydrolipoamide dehydrogenase基因群組亦全部觀察到有增加表現的現象，初步推測此基因群組除在OPEOn為唯一碳源的表現，除了能增加能量的產生外，dihydrolipoamide dehydrogenase亦能夠利用似Fenton反應產生氫氧自由基，進而藉由氫氧自由基切斷OPEOn的聚氧乙基酸鏈，此外，自由基存在量增加時，本研究進一步發現superoxide dismutase基因的表現同時被誘發，推測除了能抵抗逆境外，文獻彙整顯示尚具有切斷由醇基轉化為羧酸的功能，故其增生表現可能同時會參與OPEOn的代謝。 The octylphenol polyethoxylates (OPEOn) are recalcitrant non-ionic surfactants. The release of OPEOn has resulted in concern due to their metabolites in the environment showing estrogenic activity. However, little is known about the enzymes involved in the degradation of OPEOn. A Gram-negative rod, Pseudomonas nitroreducens TX1, was shown to be able to grow on 0.05-20% (v/v) of OPEOn as sole carbon source. Previous study also found several proteins to be up-regulated and down-regulated in response to OPEOn catabolism by 1D- and 2D-electrophoresis analysis. Consequently, this genomics study was aimed to confirm the expression of up-regulated and down-regulated proteins from functional proteomics as mentioned above. The genes in response to OPEOn catabolism and stress responses were cloned by specific primers, which were designed from reference Pseudomonas species with whole genome sequencing. The gene expression was also investigated by RT-PCR to understand if the expression of mRNA from these genes were consistent to that of proteins from 1D- and 2D- electrophoresis analysis. One gene with full length sequence and seven genes with partial sequence have been successfully cloned in this study. These genes included three transporters genes (arginine / ornithine transport system substrate-binding protein, periplasmic binding protein of ATP binding cassette transporter and TolB protein ), three genes involved in TCA cycle (2-oxoglutarate dehydrogenase, dihydrolipoamide succinyltransferase and dihydrolipoamide dehydrogenase), one gene for stress response and one gene related to fatty acid synthesis. The RT-PCR analysis indicated that the expression of these genes was consistent to that of proteins by 1D- and 2D- electrophoresis analysis except that two transporter genes, arginine / ornithine transport system substrate-binding protein and periplasmic binding protein of ATP binding cassette transporter. The expression of transporter genes found to be up-regulated only for TolB, which is related the transportation of biopolymers. The expression of dihydrolipoamide dehydrogenase gene clusters was also observed to be up-regulated. These were suggested to increase the synthesis of energy in response to OPEOn catabolism. The E3 component of these gene clusters was further proposed to generate OH radicals by Fenton-like reaction, which was demonstrated to be able to cleave the polyethoxylates in OPEOn. Moreover, the expression of superoxide dismutase was also found to be induced in response to the production of OH radicals. Since superoxide dismutase has been found to have another function for ether bond-splitting of carboxylated polyethoxylates from literature survey, the expression of this enzyme was also suggested to be involved in OPEOn catabolism.
