摘要(英) |
Alkylphenol polyethoxylates (APEOn) are non-ionic surfactants and extensively used in industrial, agricultural, and household activities. APEOn includes octylphenol polyethoxylates (OPEOn) and nonylphenol polyethoxylates (NPEOn). Large quantities of surfactants are released into environment and formed short chain APEOn (n≦3)and alkylphenol (AP). These metabolites mimic estrogenic activity, as environmental hormones, which are harmful to aquatic organisms and human. The biodegradation pathway and fate of these compounds in the environment and in organisms still remains unclear. Pseudomonas putida TX2 was isolated in Taiwan and was able to grow on 0.05~0.5% octylphenol polyethoxylates (OPEOn) or 0.02% octylphenol (OP) as sole carbon source. According to the Liquid Chromatograph-Mass Spectrometry analysis, P. putida TX2 was capable of degrading OPEOn to form OPEOn (n≦3). It also transformed OP to form octylcatechol (OC) through a sequential cleavage of ethoxylate chain. Since P. putida TX2 can grow on OP as sole carbon source, it can be a model organism to study the degradation mechanism of estrogenic-like AP. The purpose of this research is to characterize the first enzyme react on OPEOn in P. putida TX2. Using crude extract as our sample, an alcohol dehydrogenase (ADH) was found to be 48% of activity in the crude extract, 3% in periplasm and 16% of activity in the fraction between crude extract and membrane after an ultra-speed centrifugation. ADH from P. putida TX2 showed highest activity in 100 mM citric acid phosphate buffer, pH 7 at 40oC and is stable in pH 6 and 8 100 mM citric acid phosphate buffers for 42 hours. Using different enzyme assays to detect different cofactors involved in ADH, the ADH has activity toward OPEOn is a PQQ-linked and heme c containing ADH. 595 μg of protein in crude extract, ADH has 6.5, 5.2 and 5.6 mU/mg activity toward OPEOn, NPEOn, AEO8 and nonyl aldehyde, respectively and no activity to polyethylene glycol (PEG), methanol, butanol, benzyl alcohol, formaldehyde and benzaldehyde. By ionic exchange chromatography and hydrophobic interaction chromatography, there are at least two different PQQ-linked ADHs, pI<6.5 and pI>8. The first purification is a hydrophobic interaction chromatography because of the higher recovery and purification fold compared to anionic and cationic exchange chromatographies. The partially purified ADH (pI>6.5) by ionic exchange chromatographies has 5.53 mU/mg, 5.22 mU/mg and 5.07 mU/mg activity toward OPEOn, NPEOn and AEO8, respectively. It also has 7.63 mU/mg of activity toward nonyl aldehyde. No activity on PEG, methanol, butanol, benzyl alcohol, formaldehyde and benzaldehyde were detected .Therefore, we proposed that ADH (pI>6.5) in P. putida TX2 is the first to oxidize the terminal OH group of OPEOn to aldehyde then to carboxylic acid to form octylphenol polyethoxycarboxylate (OPECn). For the future work, we will clone this ADH to confirm the role in the OPEOn degradation pathway by P. putida TX2. |
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