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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/6336


    Title: 以功能性蛋白質體學研究Pseudomonas nitroreducens TX1生長於辛基苯酚聚氧乙基醇之代謝與逆境反應;Functional proteomic study on the metabolism and stress response of Pseudomonas nitroreducens TX1 grown on octylphenyl polyethoxylates
    Authors: 邱凡峰;Fang-Feng Chiu
    Contributors: 生命科學研究所
    Keywords: 辛基苯酚聚乙氧基醇;界面活性劑;次蛋白質體;鹼性區域二維電泳;蛋白質體學;octylphenol polyethoxylates;surfactant;sub-proteome;alkaline 2D;proteomics
    Date: 2005-01-07
    Issue Date: 2009-09-22 10:17:48 (UTC+8)
    Publisher: 國立中央大學圖書館
    Abstract: 辛基苯酚聚乙氧基醇(octylphenol polyethoxylates, OPEOn)為一非離子性之界面活性劑,鑑於此界面活性劑在環境中不易被微生物完全分解,且對於其代謝的機轉與途徑的了解非常有限,故本研究之目的為探討一株OPEOn分解菌(Pseudomonas nitroreducens TX1)異化界面活性劑和逆境反應。當此菌以0.5% OPEOn為唯一碳源生長培養,利用管柱層析法分離細胞粗萃液,以會對OPEOn產生耗氧活性之分液為次蛋白質體,再以一維和二維膠體電泳,分析P. nitroreducens TX1分別生長於OPEOn和succinate兩種碳源中所增生(up-regulation)或減少(down-regulation)之蛋白質,續以MALDI-Q-TOF及ESI-MS/MS鑑定之。P. nitroreducens TX1以0.5% OPEOn 為唯一碳源生長,而增強表現的蛋白質共計85個,被抑制表現的蛋白質有35個。其中,細菌外膜上的porin (Omp、OrpF與OmpH)被增生表現,而在periplasm中計有5個ABC運輸蛋白質(ATP binding cassette transporters)及1個運送生物高聚物(biopolymer)之TolB蛋白質的表現皆被增生,是否與此菌株運送OPEOn有關,需再驗證。此外,periplasm中quinoprotein ethanol dehydrogenase及aldehyde dehydrogenase亦增強表現,故推測OPEOn之聚乙氧基醇末端之氫氧基可能藉此兩個酵素先進行羧酸化,再進一步被切斷並形成acetate或是glyoxylate,但需再驗證。由於在三羧酸循環(TCA cycle)中之2-oxo-glutarate dehydrogenase (E1和E3)及succinyl-CoA synthetase皆被增生,且與氮代謝有關的ornithine carbamoyltransferase、argininosuccinate lyase及ornithine decarboxylase的表現皆增加,其中argininosuccinate lyase會裂解argininosuccinate而產生fumarate,以進入三羧酸循環加以利用,更加證實前述中TCA循環增強之推論。而利用acetyl-CoA為原料合成脂肪酸及某些胺基酸(如Cystein、Serine、Lysine)之部分酵素的表現被抑制,顯示同化作用被抑制,而acetyl-CoA 應會被驅動至三羧酸循環而生成還原能(reducing power)。本研究同時發現呼吸鏈中之NADH oxidoreductase (含FMN oxidoreductase)、electron transfer flavoprotein、Azurin及ATP synthase等表現亦增加,故推測上述TCA循環所生成的還原能,可經由電子傳遞鏈產生大量ATP供細胞利用或抗壓。此結果與本研究室先前發現P. nitroreucens TX1生長在0.5% OPEOn時氧氣消耗比生長於同濃度之succinate會增加3.7倍相符。又參與乙醛酸循環(Glyoxylate cycle)中之isocitrate lyase與malate synthase的表現增加,顯示細菌之醣質新生(gluconeogensis)可能由乙醛酸經glycerate pathway生成。而且,已知acetyl-CoA與NADH的增生會負回饋抑制pyruvate dehydrogenase,此亦與本研究發現Entner-Doudoroff pathway中,phosphoenolpyruvate synthase與pyruvate dehydrogenase (E1)之表現受抑制的結果符合。另外,逆境蛋白質亦被大量誘發表現,主要包括有過氧化逆境蛋白如alkyl hydroperoxide reductase (AhpC)、tellurium resistance protein (TerZ、TerE)、peroxidase及superoxide dismutase (SodB)與熱休克蛋白(GroEL、IbpA與DnaK)、ATP-dependent Clp protease binding protein (ClpB)等兩大類,過氧化逆境蛋白質保護細胞對抗過氧化之傷害,而熱休克蛋白與ClpB有協助蛋白質摺疊,並避免錯誤摺疊造成細胞傷害。另一調節控細胞鉀離子濃度之two-componet response regulator (KdpE)被增加表現,推測與維持菌體抵抗界面活性劑之環境壓力。整體而言,本研究利用蛋白質體學發現,P. nitroreducens TX1於界面活性劑存在的逆境下會誘發各種異化酵素、傳輸蛋白質以生成能量及多種常見於抗氧化和熱休克之逆境蛋白質,同時同化作用中脂肪酸與胺基酸的生成被抑制,以維持細胞存活與抗壓。 Octylphenol polyethoxylates (OPEOn) belongs to a nonionic surfactant family, alkylphenol polyethoxylates, which are difficult be to completely degrade in the environment. Nevertheless, the mechanism for degradation remains unclear. A Gram-nagative rod, Pseudomonas nitroreducens TX1, was previously isolated from contaminated sediment. The bacterium was able to grow on 0.05% ~20% OPEOn (Triton X-100, average n=9.5) as sole carbon source and energy. This study was aimed to investigate how P. nitroreducens TX1 metabolizes surfactant by a functional proteomics approach. OPEOn-dependent oxygen consumption activity was induced by the bacterium when grown on OPEOn as sole carbon source compared to grown on succinate. Oxygen consumption sub-proteomes were obtained from the bacterial crude extract separated by DEAE-Sepharose chromatography. Three sub-proteomes were subjected to protein by both 1D- and 2D-PAGE, and the protein spots with different expression between OPEOn- and succinate-grown sub-protemes were focused. Eighty-five up-regulated proteins and thirty-five down-regulated proteins were identified by MALDI-Q-TOF or ESI-MS/MS. The expression of outer membrane protein (porin), OrpF and OmpH, were up-regulated. Two type of transpoters, periplasmic binding proteins in ATP binding cassette transporters for transportation of polar and branch amino acids and TolB for transporting of biopolymer, were up-regulated as well. Another two periplasmic enzymes, quinoprotein ethanol dehydrogenase and aldehyde dehydrogenase, were up-regulated. The two enzymes might be related to the carboxylation of the hydroxyl terminus of the OPEOn, which was previously observed in the transformation products by strain TX1. The catabolism of OPEOn by strain TX1 was proposed by a sequential cleavage of two-carbon unit from the carboxylated ethoxylate chain to form acetate or glyoxylate. Three enzymes participated in TCA cycle, 2-oxo-glutarate dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3) and succinyl-CoA synthetase, were up-regulation. In addition, three enzymes in nitrogen metabolism, ornithine carbamoyltransferase, argininosuccinate lyase and ornithine decarboxylase, were up-regulated to form furmarate in TCA cycle. The enzymes related to the synthesis of fatty acids and some amino acids (cystein, serine, and lysine) from acetyl-CoA were down-regulation. Therefore, the increased acetyl-CoA was proposed to be driven into TCA cycle to produce reducing power. Proteins in respiratory chain, such as NADH oxidoreductase (including FMN oxidoreductase), electron transfer flavoprotein, Azurin, and ATP synthase, were also up-regulated for ATP synthesis. In our previous result, the oxygen consumption activity was induced correlating to the concentration of OPEOn (0.005~0.5%) in the growth media. Therefore, from our proteomics study, the surfactant up-regulated proteins might explain the induced OPEOn-dependent oxygen consumption in whole-cell grown on OPEOn. The enzymes in glyoxylate cycle, isocitrate lyase and malate synthase, were up-regulated, indicating the gluconeogenesis might be from glyoxlate via glycerate pathway. Generally, the increased concentration of acetyl-CoA and NADH will negatively feedback to inhibit pyruvate dehydrogenase. Therefore, we also found that the enzymes in Entner-Doudoff pathway, phosphoenolpyruvate synthase and pyruvate dehydrogenase, were observed to be down-regulated. In addition, surfactant also induced stress responsive proteins such as oxidative stress proteins, alkyl hydroperoxide reductase (AhpC), tellurium resistance protein (TerZ, TerE), peroxidase and superoxide dismutase (SodB). Other stress responsive proteins such as chaperones to mediate protein folding, heat-Shock protein (GroEL, IbpA, and DnaK), and ATP-dependent Clp protease binding protein (ClpB) were also induced. The up regulated stress responsive proteins might indicate important roles on anti-surfactant stresses. The two componet response regulator (KdpE) was up-regulated, which was proposed to be involved in the osmosis regulation. In conclusion, while OPEOn was provided to P. nitreducens TX1 as sole source of carbon and energy, the bacterium increases the expression of enzymes in catabolism and reduced those in anabolism to respond. The energy was much increased through such pathway changes. In addition, a series of anti-stress proteins were up-regulated for the survival in such a stress environment.
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