| 摘要: | 烷基苯酚聚氧乙基醇 (APEOs) 為非離子性界面活性劑界面活性劑，應用於家庭及工業用途極廣。其中Triton X-100屬於octylphenol polyethoxylate (OPEOn) 的，其平均值n為9.5，而其中間代謝物如OPEOn (n=1~3) 或是octylphenol，具環境賀爾蒙活性，已證實會累積在環境中並危害人體健康。本研究室先前自中央大學宿舍污水排放口之底泥中，篩選出可快速利用Triton X-100為唯一碳源而生長之分解菌株Pseudomonas nitroreducens TX1。本研究乃利用二維蛋白質電泳法 (pI範圍為4-8) 分析Pseudomonas nitroreducens TX1生長在0.5%或20% Triton X-100為唯一碳源之下所產生之蛋白質體，分析表現量增加強與抑制之一系列蛋白質並探討其代謝反應。研究結果所示，當Pseudomonas nitroreducens TX1生長於含0.5% succinate為唯一碳源的minimal salts basal (MSB) 培養基為對照組時，在等電點pI 4-8之間呈現的蛋白質點平均有600個；在以0.5%的Triton X-100為唯一碳源時，則可分離平均500蛋白質點：而在以20%的Triton X-100為唯一碳源時，可分辨到約600個蛋白質點。續以介質輔助雷射離子化飛行時間串聯四極質譜儀 (MALDI-Q-TOF) 鑑定Pseudomonas nitroreducens TX1的蛋白質體發現，於0.5% Triton X-100生長環境中計有17個蛋白質表現增加、11個蛋白質表現降低。其中，表現增加的蛋白質有6個屬於ABC (ATP binding cassette transporter) 運輸蛋白質，其中包括運輸支鏈胺基酸與運輸極性胺基酸兩大類，推測其中屬Leu/ Ile/ Val-binding protein family的ABC 運輸蛋白質可能與Triton X-100的輸入有關。在抗逆境反應方面，發現此菌增加表現了GroEL、ClpB、IbpA等維持蛋白質構型的抗逆境蛋白質。同時亦發現具有還原烷基過氧化物能力的AhpC蛋白質，此為第一次在界面活性壓力中被發現。加上抗鍗蛋白TerD、TerE表現量的增加，推測Triton X-100對菌體所造成的逆境與過氧化物造成的逆境相近。在滲透壓調節方面，本研究發現鉀離子的運輸蛋白KdpD與KdpE表現增加，而與滲透壓有關的調節蛋白OmpR (藉由OmpC、OmpF調整滲透壓) 則被抑制，推測此菌可能存在與Triton X-100相關的滲透壓調節系統。在能量的生成方面，dihydrolipoamide dehydrogenase在Triton X-100逆境下表現增加，此酵素參與pyruvate形成acetyl-CoA的反應，所生成acetyl-CoA進入三羧酸循環並產生能量。另一方面，其他如酯肪酸生合成蛋白質、DNA的合成蛋白質、胺基酸合成相關蛋白質則表現被抑制。本研究進一步探討此菌生長於20% Triton X-100環境中的蛋白質體，相對於0.5% Triton X-100的蛋白質體發現，ATP生合成酶、鞭毛蛋白、與酯肪酸的生合成相關蛋白等細胞周邊蛋白質的表現有增加的現象。而關於Triton X-100的代謝方面，由於乙醛酸循環中的重要酵素malate synthetase被抑制，則推測此菌切斷Triton X-100聚氧乙基醇鏈之代謝產物，以乙醛酸進入三羧酸循環的假設之可能性較低，然而已有研究發現乙醛會抑制PhoP蛋白質的表現，而本實驗亦另外觀察到PhoP蛋白質的表現被抑制，故聚氧乙基醇鏈形成乙醛而進一步被利用的可能性則較高。 Alkylphenol polyethoxylates (APEOs) are non-ionic surfactants that have been used in the household and as industrial detergents for more than 40 years. Triton X-100 belongs to the octylphenol polyethoxylates, and their metabolism intermediates such as octylphenol and octyl phenol monoethoxylate have been proved the environmental hormone. A bacterial strain, Pseudomonas nitroreducens TX1 was previously isolated from the sediments of drainage near the dormitory of National Central University in Taiwan. The bacterial strain is able to use Triton X-100 as the sole carbon and energy source to grow. This study was aimed to use 2D gel electrophoresis-based proteomic approach to identify proteins up- and down-regulated by 0.5% and 20% Triton X-100 in P. nitroreducens TX1, respectively. Results showed that there are about 500 protein spots from the pI 4-7 gels for P. nitroreducens TX1 grown under minimal salts basal medium (MSB) containing 0.5% Triton X-100 (experiment group) and about 600 spots for cells grown under 0.5% succinate as the sole carbon source (control group). 600 spots for cells grown under 20% Triton X-100 as the sole carbon source (experiment group). Moreover, the matrix-assisted laser desorption ionization-quadruples-time of fly mass spectrometer (MALDI-Q-TOF MS) was used to identify the proteins separated by two dimension gel electrophoresis (2DE). In the case of 0.5% Triton X-100, there are 17 up-regulated proteins and 11 down-regulated proteins were identified. Among the up-regulated proteins, several classes of proteins were found: (1) Six proteins were found putative ATP binding cassette (ABC) transporters, including two main classes of ABC transporters. One of the classes, the Leu/ Ile/ Val-binding protein family was suggested to involve in the transportation of Triton X-100; (2) Three were found the stress-responsive proteins (GroEL, ClpB, IbpA), which perform the function of chaperone meditating protein folding; (3) The alkyl hydroperoxide reductase (AhpC), which was first discovered in surfactant stress response; (4) Other stress proteins (TerD, TerE), which are presumed to relate to oxidative stress; (5) The K+ transporters KdpD and KdpE are highly expressed while the osmotic regulator OmpR was depressed, which suggest there may exist osmotic control system in the presence of Triton X-100. (6) One of the up-regulated enzyme is dihydrolipoamide dehydrogenase. It is related to the energy synthesis and catalyze the transformation of acetyl CoA from pyruvate. On the other hand, down-regulated enzymes are involved in the fatty acid synthesis, DNA biosynthesis and serine synthesis. The proteins up-regulated by 20% Triton X-100 were further studied in comparison to 0.5% Triton X-100. Up-regulated proteins included ATP synthetase, flagellin and fatty synthesis related proteins. Malate synthetase, one of key enzyme involved in the glyoxylate cycle, which catalyzes glyoxylate to malate was down-regulated, These suggested the degradation of polyethoxylate chain in Triton X-100 is less likely to generate glyoxylate. However, study founded acetaldehyde inhibit the expression of PhoP protein, which familiar to our experiment. It suggested the degradation of polyethoxylate side chain in Triton X-100 is more likely to from acetaldehyde. |
