Triton X-100加氧酵素之純化與定性;The purification of Triton X-100 oxygenase

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題名:	Triton X-100加氧酵素之純化與定性;The purification of Triton X-100 oxygenase
作者:	郭銀杰;Yin-Chieh Kuo
貢獻者:	生命科學研究所
關鍵詞:	加氧酵素;Triton X-100
日期:	2002-07-02
上傳時間:	2009-09-22 10:16:44 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	Triton X-100的化學結構為苯環上的酚基在其對位上接一個八個碳的支鏈烷基及平均9.5個單位的聚氧乙烯鏈混合物。此類界面活性劑被廣泛應用於農業、工業以及一般家庭使用，除此之外對於環境中有原油污染時，亦會使用大量Triton X-100將其移除，因此這類物質便會殘留於環境中；會影響土壤中有機污染物的吸附與分佈，且其本身被分解至剩1~2個氧乙烯鏈時，在結構上類似雌激素，具環境荷爾蒙效力，會影響生物體內分泌系統作用。本實驗室從土壤中篩選出，依其在不同受質，Triton X-100、dodecyl octaethoxylate（AEO8）、octyl phenol、catechol與toluene下耗氧活性從37株菌中選擇1株了同時具有Triton X-100、dodecyl octaethoxylate（AEO8）耗氧活性的Pseudomonas sp.（82.10.6）定名為Pseudomonas sp. SH4，利用耗氧儀與LC/MS的研究結果發現Triton X-100在被Pseudomonas sp. SH4分解時主要從聚氧乙烯鏈一個個切除，且NADPH參與酵素反應，我們推測它是一個NADPH-dependent oxygenase；我們經過DEAE-Sepharose、硫酸銨沉澱、Pheny-Sepharose、Mono Q and gel filtraction等純化步驟，獲得一個分子量44 kD的蛋白質，但由於N端胺基酸遇到阻礙無法進行Edman反應，因此利用胰蛋白?將蛋白質切割後經Q-ToF質譜儀分析獲得胜?質量片段與部分胺基酸序列，進入資料庫比對後結果為elongation factor Tu (EF-Tu)；EF-Tu在轉譯作用上研究很多，主要是促使aminoacyl-tRNA進入核醣體中的A區形成胜?鏈，而EF-Tu則利用EF-Ts將所帶的GDP與GTP置換，再與另一個帶有胺基酸的tRNA結合再次形成一個新的複合物，週而復始；但EF-Tu占微生物中全部蛋白的5~10 %，這麼多的蛋白質單單只進行轉譯作用似乎有點不合理，因此推測應該還有其他的功能(Kurland, et al., 1995)，最近的研究發現，EF-Tu具有在逆境下重新摺疊蛋白質或使蛋白質復性類似chaperon的功能；在滲透壓的逆境下，EF-Tu與thioredoxin有類似功能；在玉米抗熱反應中，也發現EF-Tu有保護其他蛋白質的功能；因此我們所獲得之Triton X-100 oxygenase是否即是EF-Tu需大量純化此酵素，獲得更多序列與生化的特性研究後才能確認。 Triton X-100, octylphenol polyethoxylates, belongs to nonionic surfactant. Alkyl phenol polyethoxylates are used in numberous commercial and industrical products including detergent, dispersants and emalsifiers. Large quantity of surfactants released to the enviroment result in the influence of the fate of organic compound in soils. The degraded products, alkyl phenol derivative, were demonstrated environmental hormone effect. Our previous result suguessed that the degradation of this compound by a bacteria isolated by our lab is by a sequential cleavage of the ethoxylate chain. The enzyme activity involved in the scission of ethoxylate chain was monitored by oxygen uptake rate. The ether cleavage enzyme was demonstrated as a NADPH-dependent oxygenase. The purification of the bound enzyme was carried out by DEAE-Sepharose, ammonium sulfate precipitate, Phenyl-Sepharose, MonoQ. The molecular weight of purified enzyme was 44 kD,analyzed by SDS-PAGE. The N-terminal amino acid was blocked. Therefore a trypsin-digested peptide were sequenced by Q-ToF MS. The two peptides sequences matched to an elongation factor Tu (EF-Tu). EF-Tu has been studied for many year in translation. It promotes aminoacyl-tRNA into the ribosomal A site from an EF-Tu· GTP· aminoacyl-tRNA complex with hydrolysis of GTP. The 5-10 % of total cell protein is EF-Tu and the excess over the other proteins may have other function (Kurland, et al., 1995). The recent studies has been shown that this protein may function as chaperon and thioredoxin. It plays a role in the protection of other protein from heat and osmotic stress. Whether this protein show an enzymatic activity in the oxygenation of oxygen into Triton X-100 needs to be confirmed.
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