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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)
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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
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指導教授 黃雪莉(Shir-Ly Huang) 審核日期 2010-3-2
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