Pseudomonas putida TX2 生長於辛/壬基苯酚之動力學及其鄰苯二酚加氧酵素之表現

The growth kinetics and analysis of catechol 2,3-dioxygenas(C23O)of Pseudomonas putida TX2 grows in octylphenol/nonylphenol

陳美君(Mei-chun Chen)

指導教授 ： 黃雪莉

國立中央大學/理學院/生命科學系/碩士(2008年)

摘要

烷基苯酚聚氧乙基醇類(Alkylphenyl polyethoxylates, APEOn)為非離子性的界面活性劑，已廣泛使用在工業、農業及家用;烷基苯酚(Alkylphenol, AP)為APEOn重要的中間代謝產物，一旦進入環境中，因其具雌激素活性會累積於生物體中，對於人類健康和環境生態造成危害，而在哺乳類動物中之代謝仍不明確，環境中此類化合物的生物分解主要以細菌為主。本研究篩選出以辛基苯酚(Octylphenol, OP)為唯一生長碳源之兩株菌，利用呼吸鏈指紋及16S rDNA序列分析法鑑定出兩株分別為Pseudomonas rhizosphaerae 及 Methylobacterium organophilum。將此兩株菌與本研究群先前篩出之OP分解菌Pseudomonas putida TX2和Pseudomonas sp. OP1比較，顯示P. putida TX2利用OP之生長速率，優於其他三者。P. putida TX2 來自經常施用同類界面活性機之農用土壤所製成之土壤縮模，於縮模中外加OPEOn，經兩個月馴化篩選而得，其不僅可利用寬廣濃度範圍(0.05~20%)之OPEOn，為唯一碳源生長，在最適濃度中(0.5 %)比生長速率為0.5 h-1，亦能利用(0.00025~0.1%) OP為唯一生長碳源，在最適濃度(0.002%)下比生長速率為0.47 d-1，
本研究集中在P. putida TX2利用不同碳源之生長動力學，並初步分析可能參與OP苯環切割之酵素。在生長動力學研究方面，P. putida TX2分別以各種濃度的succinate、OP、壬基苯酚 (Nonylphenol, NP)為單一碳源，或是兩兩碳源組合，利用酵素動力學相同的模式，推導比生長率(μ)、最大比生長率(μmax)、半飽和係數(KS) 、基質抑制係數(KI)等各項參數，結果顯示P. putida TX2培養在單一碳源succinate、NP、OP時，μmax分別為0.860、0.086和0.021 hr-1。Ks分別為155.41 mg/L、80.35 mg/L和122.00 mg/L。
P. putida TX2利用兩兩碳源的組合時，當高濃度succinate存在下(100~5000 mg/L)加入10~2500 mg/L OP，對菌株到達生長停滯期之最高生長量及μ沒有顯著影響，而當低濃度succinate存在下(1~50 mg/L)，加入OP則菌株最高生長量及μ隨著OP濃度上升而提高，但是當OP濃度高達5000 mg/L，則會使菌株最高生長量及μ降低。當兩種碳源組合為succinate加NP時，在1~5000 mg/L succinate存在下，菌株最高生長量及μ隨著NP濃度上升而提高，但是當NP濃度超過500 mg/L，則會使菌株最高生長量及μ降低。當菌株利用NP和OP兩種碳源的組合時，菌株最高生長量及μ均隨著NP及OP濃度上升而提高，但當OP濃度超過2500 mg/L以及NP濃度超過500 mg/L則會使菌株最高生長量及μ降低。其後利用動力學模式模擬生長情形，其結果顯示當利用succinate、NP為單一碳源時，較符合Monod equation 模式，OP為單一碳源則較符合Haldance-Andrews模式(高濃度基質抑制性)，在雙碳源部份，succinate + NP組合及succinate + OP組合，在特定濃度內，較符合雙基質無相互作用模式(Two substrate no-interaction model)，而OP + NP組合在本研究中並無與模式相符合之結果，因而顯示，本研究所面對之化合物與一般單環芳香族化合物，如甲苯等，有不同之生長競爭與抑制模式，其確切生長模式，有待更進一步探討。
本研究第二部分為對可能分解OP之酵素做初步研究，經液相層析質譜儀分析P. putida TX2之代謝產物，之前發現菌株在分解OP或NP時，偵測到烷基鄰苯二酚(Octylcatechol, OC)的生成，若再經由開苯環酵素開環，如catechol dioxygenase，則可進而被菌株利用生長。以來自P. putida mt-2之catechol 2,3-dioxygenase (C23O)所製作的抗體，經西方墨點法，分析P. putida TX2中C23O的表現;當P. putida TX2培養於0.5% succinate、0.5% OPEOn、0.005% OP、0.005% NP為唯一碳源時，皆偵測出兩種C23O (分子量分別為35 kDa及48 kDa)，進ㄧ步分析生長於不同碳源之菌體粗萃取液中之C23O酵素最高之活性;若以succinate為碳源在細胞粗萃取液之C23O對catechol之活性最高(每毫克細胞粗萃取液之蛋白質中，活性為20.1mU)，以OP為碳源時，對4-t-butylcatechol (4-t-BC)及4-t-octylcatechol (4-t-OC)有最高活性，其值同為5.0 mU/mg，若以NP為碳源生長，細菌粗萃液中之C23O對4-t-BC及4-t-OC具最高活性 (4.3 mU /mg)，推知當P. putida TX2培養於OP及NP時，菌體內被誘導出對含有中鏈和長鏈alkyl phenol之環境荷爾蒙具高專一性的開環酵素。

關鍵字

環境賀爾蒙 ； 壬基苯酚 ； 辛基苯酚 ； 生長動力學 ； 鄰苯二酚加氧酵素

並列摘要

Alkylphenyl polyethoxylates (APEOn) are non-ionic surfactants extensively used in the industrial, agricultural and household activities. Alkylphenyls including nonylphenol and octylphenol are metabolites from APEOn. They have been demonstrated as estrogenic-like environmental hormones, which are dangerous to human health and ecology. The metabolism of APEOn and AP in human is still unclear. The biodegradation of these surfactants in the environment and waste water treatment plant is mainly by bacteria. In this study, two bacterial strains were isolated and grown on octylphenol (OP) as sole carbon source. Using breath printing and 16S rDNA sequence analysis, two bacterial strains were identified as Pseudomonas rhizosphaerae and Methylobacterium organophilum. The growth rate on OP among a total of four isolates (two were previously isolates, P. putida TX2 and Pseudomonas sp. OP1) were compared. P. putida TX2 was the fastest grower on 0.005% OP. P. putida TX2 was previously isolated from a microcosm composed of made by farm soil after the exogenous addition of OPEOn for 2 months. The strain was shown to have a novel activity to grow on a wide range (0.05~20%) of OPEOn and the specific growth rate is 0.5 h-1 in 0.5% OPEOn. The strain also used 0.002% OP as sole carbon source, and the specific growth rate is 0.47 d-1.
For growth kinetics analysis, P. putida TX2 was grown in different concentrations of succinate, octylphenol and nonylphenol (NP) as a single carbon source or combination of either two, to obtain the maximum specific growth rate (μmax), half-velocity constant (KS) and substrate inhibition constant (KI). The maximum specific growth rates (μmax) are 0.86, 0.086 and 0.021 hr-1 on succinate, OP and NP, respectively. The half-velocity constants (KS) are 155.4, 80.4, and 122.0, respectively. When P. putida TX2 was grown on 100~5000 mg/L succinate plus 10~2500 mg/L OP, OP has no effect on the specific growth rate and final growth (Using OD600 from stationary phase). As OP concentration increased, the final growth and μ were also increased in the media containing 1~50 mg/L succinate. However, the specific growth rate and final growth were decreased when the OP concentration was up to 5000 mg/L. When 1~5000 mg/L succinate included in the medium, the specific growth rate and final growth of P. putida TX2 were increased according to NP concentration increasing. The specific growth rate and final growth were decreased when the NP concentration is up to than 500 mg/L. When the strain used combination of OP and NP as carbon sources, the specific growth rate and final growth were increased as NP and OP concentration increasing, but it has decreased on the specific growth rate and the final growth when the NP concentration higher than 2500 mg/L and OP concentration higher than 500 mg/L. Using the models of enzyme kinetics to study bacterial growth, succinate or NP as a single carbon source, is consistent with the Monod model and OP is with the Haldane model (inhibition resulted in higher substrate concentrations). Combination of two carbon sources, succinate plus NP or succinate plus OP, under particular concentrations, two substrates-no interaction model is applied. But there is no model to fit the results from OP plus NP data. Therefore, OP and NP are not the same as other aromatic compounds like toluene in terms of growth competition or inhibition model. Their propriate growth model remains to be further studied.
From the LC-MS analysis of the whole-cell transformation products by P. putida TX2, OP is likely to form octylcatechol (OC) and Aromatic ring-cleaved metabolites. Therefore the degraded products can be used by P. putida TX2 for growth. By using antibody against catechol 2,3-dioxygenase (C23O) from P. putida mt-2 in a western blotting experiment, two C23O-like enzymes (35 kDa and 48 kDa) were detected in the crude extract of P. putida TX2 grown on the individual carbone source (0.5% succinate, 0.5% OPEOn, 0.005% OP or 0.005% NP). The activity of catechol 2,3-dioxygenase in the crude extracts of strain TX2 were analyzed when the cells were cultivated from different carbon sources. The C23O had the highest activity (20.1 mU per mg of protein in crude extract grown from succinate when catechol is the substrate . When P. putida TX2 is grown in OP or NP as sole carbon source, C23Os from the two crude extracts all have the highest activities to 4-t-butylcatechol and 4-t-octylcatechol (5.0 and 4.3 mU/mg, respectively). It indicates that the aromatic ring-cleavage enzyme(s) which are more specific to medium and long chain alkylphenol compounds are induced in P. putida TX2 for the catabolism of OP.

並列關鍵字

Pseudomonas putida TX2 ； growth kinetics ； C23O ； catechol 2 ； octylphenol ； 3-dioxygenas ； nonylphenol

參考文獻

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