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; Study of the genes involved in the degradation of 4-t-octylphenol in Pseudomonas sp. TX1 and Acinetobacter sp. OP5 and the properties of alkylcatechol 2, 3-dioxygenase from strain OP5

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题名:	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;Study of the genes involved in the degradation of 4-t-octylphenol in Pseudomonas sp. TX1 and Acinetobacter sp. OP5 and the properties of alkylcatechol 2, 3-dioxygenase from strain OP5
作者:	阮玉俊;Tuan,Nguyen Ngoc
贡献者:	系統生物與生物資訊研究所
关键词:	生物降解;Catechol 1, 2-dioxygenase;Catechol 2, 3-dioxygenase;Phenol hydroxygenase;Biodegradation;Catechol 1, 2-dioxygenase;Catechol 2, 3-dioxygenase;Phenol hydroxygenase
日期:	2013-07-22
上传时间:	2013-08-22 11:45:47 (UTC+8)
出版者:	國立中央大學
摘要:	長鏈之烷基酚 (C=8-12)，如辛基與壬基酚等，能長期存在於環境中，且具有類似雌激素活性，該類化合物影響人類和動物的生長、繁殖與性發育，也與人類癌症有關。環境中菌株會將辛基酚與壬基酚降解至能進入細菌中央代謝途徑之代謝物而去除。前人研究發現中、短鏈烷基酚可由數個 Pseudomonas 屬的菌株經開環途徑降解；而長鏈烷基酚則可由Sphingomonas 屬的菌株以 alkylphenol monooxygenase 經 ipso-hydroxylation 機制進行降解，其中 ipso-hydroxylation降解機制尚未被完全研究清楚。而自然環境中其他菌株之降解途徑尚不清楚，故快速篩選長鏈烷基酚降解菌株用以瞭解具雌激素活性之長碳鏈烷基酚在自然環境中的降解機制是很重要的。先前在黃雪莉教授團隊已分離出 18 株4-tert-辛基酚降解菌株，並再分離一株 Acinetobactor sp. OP5；本研究設計四組引子，經聚合?鏈鎖反應篩選菌株內具有降解功能之基因。這些引子是設計來放大以下酵素的特定片段：其中包含對苯環進行加氧作用的: (1) multicomponent phenol hydroxylase、(2) single component monooxygenase；以及對苯環進行切割的 (1) catechol 1, 2-dioxygenase、(2) catechol 2, 3-dioxygenase genes。其中 18 株菌都有一段長度 232 bp 之 multicomponent phenol hydroxylase，並由中鏈 (C=3-7) 和短鏈 (C=1-2) 之烷基酚降解菌株中分離出的 multicomponent phenol hydroxylase 有61% 至 92% 不等之相似度。而此18株中的13株均含有一段長度324 bp之catechol 1, 2-dioxygenase，與已發表且序列最接近之catechol 1, 2-dioxygenase有78% ~ 95% 之相似度。而以下三株菌同時具有multi-component phenol hydroxylase，catechol 1, 2-dioxygenase與catechol 2, 3-dioxygenase genes：Pseudomonas putida TX2、Pseudomonas sp. TX1 與 Acinetobacter sp. OP5，故本研究集中在Pseudomonas sp. TX1 與Acinetobacter sp. OP5兩株菌株的分解基因上。菌株TX1 的multicomponent phenol hydroxylase 基因簇與 catechol 2, 3-dioxygenase基因之全長已完全確認並轉殖。Acinetobacter sp. OP5 中 multicomponent phenol hydroxylase 的基因簇 (ophRBA1A2A3A4A5A6CEH) 包含調控子 (OphR)、alkylcatechol 2, 3-dioxygenase (OphB)、multicomponent phenol hydroxylase (OphA1A2A3A4A5A6)、2-hydroxymuconic semialdehyde dehydrogenase (OphC)、2-hydroxypent-2,4-dienoate hydratase (OphE), and 4-oxalocrotonate decarboxylase (OphH) 與 catechol 2, 3-dioxygenase (OphB) 的基因已被選殖，而此段基因簇對已發表之中等長度烷基酚基因群有高度的同源性。以 E. coli為宿主表達 ophB 的產物 alkylcatechol 2,3-dioxygenase 對 4-methylcatechol (1,435%)、 4-ethylcatechol (982%)、catechol (100%)、4-t-butylcatechol (16.6%)，與 4-t-octylcatechol (3.2%) 均具有meta-cleavage之活性，證實該基因對具有甲基與乙基之鄰苯二酚之活性高於無支鏈之鄰苯二酚。但對碳數在四以上之烷基鄰苯二酚，其活性則隨著碳鏈增長而降低，亦可解釋大自然環境中辛、壬基酚之不易分解且易累積之機制。本論文結果顯示發展分子技術可以有效且簡單的偵測中長鏈烷基酚之分解菌和基因群，並發現對具有雌激素活性有開環作用之酵素，而可進而破壞類雌激素活性，我們並發表第一個對高度分支的烷基酚之開環酵素。 Long-chain alkylphenols (C=8-12), such as nonylphenols and octylphenols, are estrogen-like compounds that are persistent in the environment until they are degraded by bacteria. These compounds influence the growth, reproduction and sexual development of humans and other animals, therefore are a cause of health and ecology related concerns. Some studies of the biodegradation of long-chain alkylphenols have been reported. The mechanism of few Sphingomonas strains was reported using the ipso-hydroxylation mechanism by the analysis of degradation metabolites. For many other strains, the mechanism and pathways were still unknown. Therefore, it has become important to quickly screen the diversity of long-chain alkylphenol-degrading strains and hopefully gain a better understanding of how the estrogenic activity from long-chain alkylphenols is disrupted in the natural environment. In this study, nineteen 4-t-octylphenol-degrading bacteria were previously isolated by Huang’s team. They were screened for the presence of representative degradative genes by polymerase chain reaction method using four designed primer sets. The primer sets were designed to amplify specific fragments from (1) multicomponent phenol hydroxylase, (2) single component monooxygenase, (3) catechol 1, 2-dioxygenase and (4) catechol 2, 3-dioxygenase genes. None of the isolates showed amplification of a single component monooxygenase gene. Eighteen of the nineteen isolates exhibited the presence of a 232 bp amplicon that shared 61% to 92% identity to known multicomponent phenol hydroxylase gene sequences from short (C=1-2) and/or medium-chain (C=3-7) alkylphenol-degrading strains. Thirteen of the eighteen isolates were positive for a 324 bp region that exhibited 78% to 95% identity to the closest published catechol 1, 2-dioxygenase gene sequences. Interestingly, three strains, Pseudomonas putida TX2, Pseudomonas sp. TX1 and Acinetobactter sp. OP5 revealed the presence of three genes (multicomponent phenol hydroxylase, catechol 1, 2-dioxygenase genes and catechol 2, 3-dioxygenase). Therefore, strain TX1 and OP5 were further focused for their degradation genes. For strain TX1, the complete multicomponent phenol hydroxylase and catechol 2, 3-dioxygenase genes were identified. For strain OP5, a gene cluster (ophRBA1A2A3A4A5A6CEH) encoding regulator (OphR), alkylcatechol 2, 3-dioxygenase (OphB), multicomponent phenol hydroxylase (OphA1A2A3A4A5A6), 2-hydroxymuconic semialdehyde dehydrogenase (OphC), 2-hydroxypent-2,4-dienoate hydratase (OphE), and 4-oxalocrotonate decarboxylase (OphH) was then cloned from Acinetobacter sp. strain OP5 and showed the highest homology to those involved in the published medium-chain alkylphenol gene clusters. The purified enzyme from recombinant alkylcatechol 2, 3-dioxygenase showed meta-cleavage activities for 4-methylcatechol (1,435%), 4-ethylcatechol (982%), catechol (100%), 4-t-butylcatechol (16.6%), and 4-t-octylcatechol (3.2%), demonstrating the preference on the substrate bearing methyl and ethyl group than catehol. With the increase of the length of alkylchain, the activities decrease, this demonstrated the one of the possibilities of accumulation of octylphenol or nonylphenol in the environment. This study demonstrates the first alkylcatechol 2, 3-dioxygenase which work on a highly branched alkylcatechol. Considering the results, we conclude that the developed molecular technique is useful and easy in detection of medium/long-chain alkylphenol degradation gene cluster and bacterial strains which involved in the disruption of the aromatic ring of the structure which may cause the disruption of the estrogen-like activity.
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