Pseudomonas putida TX2中辛基苯酚聚氧乙基醇類脫氫酶之初步純化與特性研究

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：15

、訪客IP：52.15.92.58

姓名

曾愛倫(Ai-lun Tseng) 查詢紙本館藏

畢業系所

系統生物與生物資訊研究所

論文名稱

Pseudomonas putida TX2中辛基苯酚聚氧乙基醇類脫氫酶之初步純化與特性研究
(Purification and characterization of alcohol dehydrogenase from Pseudomonas putida TX2)

相關論文

★ 以電泳膠體分離及質譜儀鑑定紅斑性狼瘡(活躍期)血漿中表現差異的蛋白質	★ Acinetobacter sp. OP5 與 Pseudomonas sp. TX1 參與辛基酚分解之基因群與OP5菌株之烷基鄰苯二酚2, 3加氧酵素
★ 陰離子界面活性劑sodium dodecylbenzene sulfonate分解菌篩選與脫磺酸酵素研究	★ 鄰苯二酚加氧酵素的熱穩定性提昇研究
★ Triton X-100 分解菌之分離和分解酵素之特性研究	★ Triton X-100加氧酵素之純化與定性
★ Lactobacillus reuteri於酸性與膽鹽環境中之蛋白質體研究	★ 蕃茄根部受銅逆境之基因調控
★ Pseudomonas nitroreducens TX1 異化辛基苯酚聚氧乙基醇之功能性蛋白質體學：以二維電泳法分析等電點4-8之蛋白質表現	★ Pseudomonas nitroreducens TX1之具耗氧活性之麩胺酸合成酶之單離
★ 人類細胞株生產含多種亞型的干擾素-a之蛋白質體學研究	★ 辛基苯酚之分解:分解菌和生物復育之菌相研究
★ 分解辛基苯酚聚氧乙基醇之耗氧酵素（二氫硫辛醯胺脫氫酶）的純化與定性	★ AtNPR1轉殖番茄之性狀分析及抗病機制研究
★ Pseudomonas putida TX2分解辛基苯酚聚氧乙基醇及其具雌激素活性代謝物之研究	★ 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

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

至系統瀏覽論文 ( 永不開放)

摘要(中)

烷基苯酚聚氧乙基醇 (alkylphenol polyethoxylates, APEOn) 常被用於工業，農業及一般家庭所使用的非離子界面活性劑，其中壬基苯酚聚氧乙基醇 (NPEOn) 以及辛基苯酚聚氧乙基醇 (OPEOn)最為廣泛使用。這些大量的界面活性劑排放在環境中被微生物分解成短鏈之APEOn 及烷基苯酚(alkylphenol, AP)。短鏈的APEOn 與AP 結構類似人類雌激素故會造成環境荷爾蒙效應。目前對於微生物降解烷基苯酚聚氧乙基醇之途徑並非十分清楚。本實驗室已篩選出一株可利用0.05~20%辛基苯酚聚氧乙基醇或0.02%辛基苯(octylphenol, OP)為唯一生長碳源之菌株: Pseudomonas putida TX2。根據先前液相層析質譜儀的分析，P. putida TX2 可逐步切斷OPEOn 的氧乙基鏈，生成OP 及octyl catechol。本研究主要目的即在由P. putida TX2 生長在OPEOn 時，發現醇類脫氫酶(alcohol dehydrogenase, ADH)可切斷OPEOn的氧乙基鏈。利用不同輔因子的醇類脫氫酶酵素活性法估推此作用在OPEOn 之ADH 是含有pyrroloquinoline quinone (PQQ)並稱為ADH(PQQ)。將細胞打破並離心之後，發現大部分ADH (PQQ) 之活性位於細胞粗萃取液且占總活性之48%，經疏水性管柱分液出來的部分醇化的酵素，在pH 7 及40oC 的條件下活性最高而陰離子管柱所部分純化出來的酵素在pH 8 及 45oC 的條件下活性最高。細胞粗萃取液及此部份純化的ADH (PQQ)對於Dodecyl octylethoxylate (AEO8)都具有最大的親和力其次為NPEOn 及OPEOn，而ADH (PQQ)對OPEOn 有最大的反應速率，其次為NPEOn，最後為AEO8。比較疏水性管柱層析及離子交換管柱之後可得知疏水性管柱可得到較純的酵素，所以被選為第一個純化步驟，再利用陰離子交換管柱層析，將有活性的部分分離出有吸附及沒有吸附的酵素，其大多數有ADH (PQQ)活性的酵素都位於沒有吸附的部分。將這些沒有吸附但具有ADH (PQQ)活性的酵素使用液相層析質譜儀分析，結果顯示在80 分鐘後有OPEO3 的出現及OPEOn, n = 13 及14 的消失，因此推測P. putida TX2 之ADH (PQQ)是將OPEOn 之連續氧化末端氫氧基成短鏈的氧乙基鏈。

摘要(英)

Alkylphenol polyethoxylates (APEOn), including octylphenol polyethoxylates (OPEOn) and nonylphenol polyethoxylates (NPEOn), are non-ionic surfactants and extensively used in industrial, agricultural, and household products. Large quantities of surfactants are often released into the environment and are majorly degraded by microorganisms. These degraded products APEOn, n = 0~3 and alkylphenol (AP), act as environmental hormones which mimic the estrogenic activity that are harmful to the aquatic organisms and human health. The complete metabolic pathways and degrading enzymes still remain unclear. Pseudomonas putida TX2 was isolated from a paddy field in Il-Lan, Taiwan, which shown to grow on 0.05~20% of OPEOn or 0.02% of OP as sole carbon source. From the previous studies by LC-MS analysis, P. putida TX2 was able to degrade OPEOn to OPEOn (n≦3) and the formation of octylphenol (OP) by a whole-cell transformation study. The purpose of this research is to isolate an enzyme which can oxidize OPEOn. In this study, an alcohol dehydrogenase (ADH) from P. putida TX2 was preliminarily demonstrated to react on the substrate and then was partially purified. The molecular weight of the denatured protein was estimated to be 55 kDa. When testing using different cofactors, such as NAD(P)+, FAD+ and PQQ in the enzyme assays, ADH was predicted to be a pyrroloquinoline quinone
(PQQ)-linked ADH. Most of the active ADH (PQQ) was present in the crude extracts of the cell, which takes about 48% of the total activity. Both of the ADH (PQQ) in the crude extracts and partially purified ADH (PQQ) after hydrophobic interaction chromatography showed the highest activity at pH 7 and 40oC. However, the pH and temperature are slightly higher after anionic exchange chromatography, i. e., pH 8 and 45oC, respectively. The Lineweaver-Burk plot showed all the enzymes in the crude extracts and from the partially purified ADH (PQQ) have the highest affinity toward AEO8 follow by NPEOn and OPEOn; however, the maximum rate is highest toward OPEOn to NPEOn and finally to AEO8. The hydrophobic interaction chromatography
was the first purification column used due to higher recovery and purification fold. A strong anionic exchange column was used for the next purification step, but the ADH
did not bind on the column. The catalytic products from partially purified ADH from the flow-through of the anionic exchange column were subjected onto LC-MS. The ethoxylate units of OPEOn were shorten after 80 minutes with the presence of OPEO3 and the disappearance of OPEOn, n = 13 and 14 according to the mass spectrum. Aldehyde was not detected, thus ADH in P. putida TX2 transformation is predicted to simultaneously oxidize the hydroxyl groups and aldehyde moieties (based on the substrate specificity assay) by alcohol dehydrogenase.

關鍵字(中)

★ 醇類脫氫酶
★ 辛基苯酚聚氧乙基醇
★ 酵素純化
★ 微生物降解
★ 非離子界面活性劑

關鍵字(英)

★ octylphenol polyethoxylates
★ enzyme purification
★ alcohol dehydrogenase
★ non-ionic surfactants
★ biodegradation

論文目次

Abstract ……………………………………………………………… i
Acknowledgement ………………………………………………………v
Table of content ……………………………………………………vi
List of figures………………………………………………… viii
List of tables ………………………………………………………ix
List of abbreviations …………………………………………… x Introduction …………………………………………………………1
Nonionic surfactants ………………………………………………1
Environmental Hormones ……………………………………………2
Biodegradation of alkylphenol polyethoxylates or related compounds ………………………………………………………………2
Micorbial degradation ………………………………………… 2
Enzymatic degradation ………………………………………… 4
Other APEOn degrading enzymes found in the lab ……………7
Aims of the thesis ……………………………………………… 7
Materials and methods ………………………………………………9
Chemicals …………………………………………………………… 9
Bacterial strain and cultural conditions ………………… 9
Bacterial morphology …………………………………………… 10
Enzyme preparation and quantification ………………………10
Preparation of periplasmic, cytoplasmic and membrane proteins ………………………………………………………………10
Enzyme assay ……………………………………………………… 11
Enzyme characteristic assay ……………………………………11
pH ………………………………………………………………… 11
Temperature ……………………………………………………… 11
Enzyme purification …………………………………………… 11
Phenyl superose (a hydrophobic interaction) …………… 11
Mono Q (strong anionic exchange interaction) ……………12
HiTrap SP HP (strong cationic exchange interaction) … 12
Chloroform extraction ……………………………………………13
LC-MS analysis …………………………………………………… 13
Instruments …………………………………………………………13
Results ……………………………………………………………… 15
The growth of Pseudomonas putida TX2 ……………………… 15
Sequence analysis and molecular characterization ……… 15
Alcohol dehydrogenase distribution of Pseudomonas putida TX2 …………………………………………………………………… 16
Partial purification of alcohol dehydrogenase…………… 16
Enzyme characteristics ………………………………………… 17
Pseudomonas putida TX2 transformation of OPEOn ………… 20
Discussion ……………………………………………………………21
Bacterial and enzyme properties ………………………………21
Localization of alcohol dehydrogenase ………………………22
Enzyme assay of ADH(PQQ) of Pseudomonas putida TX2 …… 23
Enzyme purification ………………………………………………24
Enzyme characteristics analysis ………………………………25
Mass analysis of partially purified ADH(PQQ) transforming OPEOn ………………………………………………………………… 25
The predicted mechanism of OPEOn degradation by ADH ……26
Conclusion ………………………………………………………… 28
Reference …………………………………………………………… 30
Tables …………………………………………………………………35
Figures ……………………………………………………………… 43
Appendix ………………………………………………………………67

參考文獻

賴建宏。2004。Pseudomonas nitroreducens TX1 之具耗氧活性之麩氨酸合成酶之單離。國立中央大學生命科學研究所碩士論文。
謝孝正。2004。Pseudomonas putida TX2 分解辛基苯酚聚氧乙基醇及具雌激素活性代謝物之研究。國立中央大學生命科學研究所碩士論文。
邱凡峰。2005。以功能性蛋白質體學研究Pseudomonas nitroreducens TX1 生長於辛基苯酚聚氧以基醇之代謝與逆境反應。國立中央大學生命科學研究所碩士論文。
鍾依靜。2008。Pseudomonas putida TX2 中催化辛基苯酚聚氧乙基醇之醇類脫氫酶的初步定性。國立中央大學生命科學研究所碩士論文。
黃雪莉。2002。界面活性劑之微生物分解。微生物資源與應用研討會論文集。p.159-175
Ameyama, M., Adachi, O., 1982. Alcohol dehydrogenase from acetic acid bacteria,membrane-bound. Methods Enzymology. 89, 450-457.
Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72, 248-54.
Brunner H., Silvio, C., Antonio, M., Walter, G., 1988. Occurrence and behavior of linear alkylbenzenesulphonates, nonylphenol, nonylphenol mono- and nonylphenol diethoxylates in sewage sludge treatment. Water Research. 22,1465-1472.
Chen, H. J., Tseng, D. H., Huang, S. L., 2005. Biodegradation of octylphenolpolyethoxylate surfactant Triton X-100 by selected microorganisms. Bioresour Technol. 96, 1483-91.31
Cozier, E., Giles, G., Anthony, C., 1995. The structure of the quinoprotein alcohol dehydrogenase of Acetobacter aceti modelled on that of methanol dehydrogenase from Methylobacterium extorquens. Biochem J. 308 ( Pt 2),375-9.
ENDS 1995. FoE seeks ban on alkyl phenols. ENDS Report 241: 11-12
Ghosh, M., Anthony, C., Harlos, K., Goodwin, G., Blake, C., 1995. The refined structure of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens at 1.94 A. Structure. 3, 177-87.
Giger, W., Brunner, H., Schaffner, C., 1984. 4-Nonylphenol in sewage sludge: accumulation of toxic metabolites from nonionic surfactants. Science. 225, 623-5.
Gomez-Manzo, S., Contreras-Zentella, M., Gonzalez-Valdez, A., Sosa-Torres, M., Arreguin-Espinoza, R., Escamilla-Marvan, E., 2008. The PQQ-alcohol dehydrogenase of Gluconacetobacter diazotrophicus. Int J Food Microbiol. 125, 71-8.
Gorisch, H., Rupp, M., 1989. Quinoprotein ethanol dehydrogenase from Pseudomonas. Antonie Van Leeuwenhoek. 56, 35-45.
Groen, B., Frank, J., Duine, A., 1984. Quinoprotein alcohol dehydrogenase from ethanol-grown Pseudomonas aeruginosaI. Biochemistry Journal. 223, 291-294.
Gross, A., Ong, R., Grant, R., Hoffmann, T., Gregory, D., Sreerama, L., 2009. Human aldehyde dehydrogenase-catalyzed oxidation of ethylene glycol ether aldehydes. Chem Biol Interact. 178, 56-63.
Hideaki, M., Masuda, N., Fujiwara, Y., Ike, M., Fujika, M., 1994. Degradation of alkylphenol ethoxylates by Pseudomonas sp. Strain TR01. Appl Environ Microbiol. 60, 2265-2271.
John, M., White, F., 1998. Mechanism for biotransformation of nonylphenol polyethoxylates to Xenoestrogens in Pseudomonas putida. J Bacteriol. 180, 4332-8.
Jongejan, A., Jongejan, A., Duine, A., 1998. Homology model of the quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni. Protein Eng. 11, 185-98. 32
Kawai, F., Yamanaka, H., Ameyama, M., Shinagawa, E., Matsushita, K., 1985. Identification of the prosthetic group and further characterization of novel enzyme, polyethylene glycol dehydrogenase. Agricultural and Biological Chemistry. 49, 1071-1076.
Liu, X., Tani, A., Kimbara, K., Kawai, F., 2007. Xenoestrogenic short ethoxy chain nonylphenol is oxidized by a flavoprotein alcohol dehydrogenase from Ensifer
sp. strain AS08. Appl Microbiol Biotechnol. 73, 1414-22.
Matsushita, K., Shinagawa, E., Adachi, O., Ameyama, M., 1982. o-Type cytochrome oxidase in the membrane of aerobically grown Pseudomonas aeruginosa. FEBS
Lett. 139, 255-8.
Meyers, D., 1992. Surfactant Science and Technology. VCH Publishers, New York, 2nd ed.
Nimrod, C., Benson, H., 1996. Environmental estrogenic effects of alkylphenol ethoxylates. Crit Rev Toxicol. 26, 335-64.
Ohta, T., Tani, A., Kimbara, K., Kawai, F., 2005. A novel nicotinoprotein aldehyde dehydrogenase involved in polyethylene glycol degradation. Appl Microbiol Biotechnol. 68, 639-46.
Pagano, M., Lopez, A., Volpe, A., Mascolo, G., Ciannarella, R., 2008. Oxidation of nonionic surfactants by Fenton and H2O2/UV processes. Environ Technol. 29,
423-33.
Reddy, Y., Bruice, C., 2004. Mechanisms of ammonia activation and ammonium ion inhibition of quinoprotein methanol dehydrogenase: a computational approach. Proc Natl Acad Sci U S A. 101, 15887-92.
Reichmann, P., Gorisch, H., 1993. Cytochrome c550 from Pseudomonas aeruginosa. Biochem J. 289 ( Pt 1), 173-8.
Reid, F., Fewson, A., 1994. Molecular characterization of microbial alcohol dehydrogenases. Crit Rev Microbiol. 20, 13-56.
Rupp, M., Gorisch, H., 1988. Purification, crystallisation and characterization of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa. Biol Chem Hoppe Seyler. 369, 431-9.
Sato, H., Shibata, A., Wang, Y., Yoshikawa, H., Tamura, H., 2003. Characterization of biodegradation intermediates of nonionic surfactants by MALDI-MS. 2.
Oxidative biodegradation profiles of uniform octylphenol polyethoxylate in 18O-labeled water. Biomacromolecules. 4, 46-51. 33
Schrover, J., Frank, J., van Wielink, E., Duine, A., 1993. Quaternary structure of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa and its reoxidation with a novel cytochrome c from this organism. Biochem. J. 290,123-127.
Tabira, Y., Nakai, M., Asai, D., Yakabe, Y., Tahara, Y., Shinmyozu, T., Noguchi, M., Takatsuki, M., Shimohigashi, Y., 1999. Structural requirements of para-alkylphenols to bind to estrogen receptor. Eur J Biochem. 262, 240-5.
Tanenbaum, M., Wang, Y., Williams, P., Sigler, B., 1998. Crystallographic comparison of the estrogen and progesterone receptor's ligand binding domains. Proc Natl
Acad Sci U S A. 95, 5998-6003.
Tasaki, Y., Yoshikawa, H., Tamura, H., 2006. Isolation and characterization of an alcohol dehydrogenase gene from the octylphenol polyethoxylate degrader Pseudomonas putida S-5. Biosci Biotechnol Biochem. 70, 1855-63.
Toyama, H., Matthews, S., Adachi, O., Matsushita, K., 2004. Quinohemoprotein alcohol dehydrogenases: structure, function, and physiology. Arch Biochem Biophys. 428, 10-21.
Xu, F., 2005. Applications of oxidoreductases: recent progress. Industrial Biotechnology. 1, 38-50.
Yamanaka, H., Kawai, F., 1989. Purification and characterization of constitutive polyethylene glycol (PEG) dehydrogenase of a PEG 4000-utilizing Flavobacterium sp. no. 203. Journal of Fermentation and Bioengineering. 67,
324-330.
Yamanaka, K., Tsuyuki, Y., 1983. A new dye-linked alcohol dehydrogenase (vanillyl alcohol dehydrogenase) from Rhodopseudomonas acidophila M402: purification, identification of reaction product and substrate specificity. Agricultural and Biological Chemistry. 47, 2173-2183.
Yamashita, M., Tani, A., Kawai, F., 2004. A new ether bond-splitting enzyme found in Gram-positive polyethylene glycol 6000-utilizing bacterium, Pseudonocardia sp. strain K1. Appl Microbiol Biotechnol. 66, 174-9.
Ying, G., Williams, B., Kookana, R., 2002. Environmental fate of alkylphenols and alkylphenol ethoxylates--a review. Environ Int. 28, 215-26.
Ying, X., Grunden, M., Nie, L., Adams, W., Ma, K., 2009. Molecular characterization of the recombinant iron-containing alcohol dehydrogenase from the hyperthermophilic Archaeon, Thermococcus strain ES1. Extremophiles. 13, 299-311. 34
Zarnt, G., Schrader, T., Andreesen, R., 1997. Degradation of tetrahydrofurfuryl alcohol by Ralstonia eutropha is initiated by an inducible pyrroloquinoline quinone-dependent alcohol dehydrogenase. Appl Environ Microbiol. 63, 4891-8.
Vazquez-Laslop, N., et al., 2001. Molecular sieve mechanism of selective release of cytoplasmic proteins by osmotically shocked Escherichia coli. J Bacteriol. 183,
2399-404.

指導教授

黃雪莉(Shir-Ly Huang)

審核日期

2010-3-2

推文