摘要(英) |
Alkylphenol polyetholates (APEOn) are nonionic surfactants that are widely used today. They are composed of a polyethoxy chain and an alkyl chain connected to a phenyl structure at para-position. In this study, an analysis method to detect microbial biodegradation products from APEOn was established. In our previous study, Pseudomonas nitroreducens TX1 can grow on octylphenol polyethoxylates (OPEOn). The strain showed OPEOn-dependent oxygen consumption activity induced when it was grown on OPEOn as sole carbon source. But the activity was not found when grown on 0.5% succinate, suggesting oxygen uptake activity is involved in the metabolism of OPEOn. By using the oxygen uptake assay, glutamate synthase was purified from this bacterial strain.
In the analysis of OPEOn metabolic products, the transformed products from two substrates (OPEOn and dodecyl octylethoxylate(AEO8)) by crude extract or partially purified enzyme were analyzed by HPLC-MS. The ethoxylate chain was shortened and AEO7 formed by crude extract, while OPEOn with shortened ethoxylate chain (n=3-12) and octylphenol polyethoxycarboxylate (OPECn, n=5-9) were the transformation products by partially purified enzyme. By gas chromatography-mass spectrometry (GC-MS), metabolites such as OPEO2, some products with the structure of polyethoxylate, and carboxylated polyethoxylate were formed by crude extract. P. nitroreducens TX1 may undergo carboxylation of terminal ethoxylate alcohol and ether chain cleavage to metabolize OPEOn and AEO8.
In the purification of enzyme related to the metabolism of OPEOn, two oxygen consumption activity areas from DEAE-Sepharose chromatography of the crude extract of strain TX1 were detected; one that could not bind to the column and the other that was eluted by 0.2-0.4 M KCl. About equal activity were recovered from this step. The second pool was further purified by Phenyl-Sepharose, Mono-Q, hydroxyapatite, and second Mono-Q chromatography. The purified enzyme was identified as glutamate synthase by peptide finger-printing from ESI-MS/MS. A molecular mass of native protein was estimated as 190 ±10 kDa by gel filtration with two subunits of 144 ±6 and 54 ±4 kDa MW respectively from SDS-PAGE. The result indicates that the enzyme was a heterodimer (?1?1). The absorption spectrum at 200-650 nm showed that the pure enzyme contains flavin (FAD or FMN) in oxidized state. However, the transformation products by the purified glutamate synthase revealed no ethoxylate chain shortage. Based on the studies by other groups, OPEOn is very unlikely to be the substrate of the enzyme. In our study, the enzyme showed oxygen uptake activity in 40 mM potassium phosphate buffer, pH 7.0, containing 0.05% OPEOn, 3 mM metal mixture, and 0.5 mM NADPH. The specific activity of oxygen uptake was 205 nmole/min/mg. When the metal mixture was replaced by either 3 mM Mn2+ or Cu2+ ion, the specific activity was increased 2-3 fold compared to that with no metal control. This is the first report of oxygen uptake activity for glutamate synthase. The role of the enzyme in OPEOn metabolism and oxygen uptake activity from OPEOn-grown bacterium needs to be further investigated.
The partial purified enzyme that showed degraded products from OPEOn (short chain OPEOn and OPECn) was purified from P. nitroreducens TX1 using DEAE-Sepharose, Phenyl-Sepharose, and Mono Q chromatography. They were composed of seven major proteins. However, after further purification, the OPEOn degrading activity was lost from the next chromatography. One of the seven proteins, ornithine carbamoyltransferase, was found to be up-regulated by OPEOn from our proteomic study. However, glutamate synthase was not found to be up-regulated. |
參考文獻 |
賴耿陽. 譯. 1994. 界面活性劑應用實務. 復漢出版. p.375-358
楊嘉蓁. 2001. TritonX-100分解菌之分離與分離酵素之特性研究. 國立中央大學生命科學研究所碩士論文.
陳怡君. 2001. 土壤對Triton系列各EO鏈選擇性吸附之研究. 國立中央大學環境工程研究所碩士論文.
郭銀杰. 2002. Triton X-100 加氧酵素之純化與定性. 國立中央大學生命科學研究所碩士論文.
洪國展. 2004. 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. 國立中央大學生命科學研究所碩士論文
Ahel M, Conrad T, Giger W. 1987. Persistent organic chemicals in sewage effluents. 3. Determinations of nonylphenoxy carboxylic acids by high-resolution gas chromatography/mass spectrometry and high-performance liquid chromatography. Environ . Sci. Technol. 21: 697-703
Ahel M, Giger W. 1985a. Determination of alkylphenols and alkylphenol mono- and diethoxylates in environmental samples by high-performance liquid chromatography. Anal. Chem. 57: 1577-1583
Ahel M, Giger W. 1985b. Determination of nonionic surfactants of the alkylphenol polyethoxylate type by high-performance liquid chromatography. Anal. Chem. 57: 2584-2590
Ahel M, Giger W, Molnar E, Ibric S. 2000. Determination of nonylphenol polyethoxyltes and their lipophilic metabolites in sewage effluents by normal-phase high-performance liquid chromatography and fluorescence detection. Croatica Chem. Acta 73: 209-227
Ahel M, Schaffner C, Giger W. 1996. Behaviour of alkylphenol polyethoxylate surfactants in the aquatic environment. 3. Occurrence and elimination of their persistent metabolities during infiltration of river water to groundwater. Water Res. 30: 37-46
Brand N, Mailhot G, Bolte M. 1998a. Degradation Photoinduced by Fe(III): Method of Alkylphenol Ethoxylates Removal in Water. Environ. Sci. Technol. 32: 2715 -2720
Brand N, Mailhot G, Bolte M. 1998b. Degradation photoinduced by Fe(III): Method of alkylphenol ethoxylates removal in water. Environ. Sci. Technol. 32: 2715-2720
Csonka LN, Epstein W. 1996. Osmoregulation In Escherichia coli and Salmonella. Cellular and Molecular Biology: 1210-1223
Di Corcia A, Cavallo, R., Crescenzi, Carlo., and Nazzari, Manuela. 2000. Occurrence and Abundance of Dicarboxylated Metabolites of Nonylphenol Polyethoxylate Surfactants in Treated Sewages. Environ. Sci. Technol. 34: 3914 -3919
Ding WH, Fujita, Y., Aeschimann, R., Reinhard, M. 1996. Identification of organic residues in tertiary effluents by GC/EI-MS, GC/CI-MS and GC/TSQ-MS. Fresenius J. Anal. Chem 354: 48-55
Ferguson PL, Iden, C. R. and Brownawell, B. J. 2001. Distribution and Fate of Neutral Alkylphenol Ethoxylate Metabolites in a Sewage-Impacted Urban Estuary. Environ. Sci. Technol. 35: 2428 -2435
Frings J, Schink B. 1994. Fermentation of phenoxyethanol to phenol and acetate by a homoacetogenic bacterium. Arch Microbiol 162: 199-204
Frings J, Schramm E, Schink B. 1992. Enzymes involved in anaerobic polyethylene glycol degradation by Pelobacter venetianus and Bacteroides strain PG1. Appl. Environ. Microbiol. 58: 2164-2167
Gundersen JL. 2001. Separation of isomers of nonylphenol and select nonylphenol polyethoxylates by high-performance liquid chromatography on a graphite carbon column. J. Chromatogr. A 914: 161-166
Hoai PM, Tsunoi S, Ike M, Kuratani Y, Kudou K, et al. 2003. Simultaneous determination of degradation products of nonylphenol polyethoxylates and their halogenated derivatives by solid-phase extraction and gas chromatography-tandem mass spectrometry after trimethylsilylation. J Chromatogr A 1020: 161-171
John DM, White GF. 1998. Mechanism for biotransformation of nonylphenol polyethoxylates to Xenoestrogens in Pseudomonas putida. J Bacteriol 180: 4332-4338
John DMaW, G.F. 1998. Mechanism for biotransformation of nonylphenol polyethoxylates to xenoestrogens in Pseudomonas putida. J Bacteriol 180: 4332-4338
Kawai F. 1985. Existence of ether bond-cleaving enzyme in polyethylene glycol-utilizing symbiotic mixed culture. FEMS Microbiol. Lett. 30: 273-276
Korner W, Spengler P, Bolz U, Schuller W, Hanf V, Metzger JW. 2001. Substances with estrogenic activity in effluents of sewage treatment plants in southwestern Germany. 2. Biological analysis. Environ Toxicol Chem 20: 2142-2151
Light DR, Walsh C, Marletta MA. 1980. Analytical and preparative high-performance liquid chromatography separation of flavin and flavin analog coenzymes. Anal Biochem 109: 87-93
Marcomini A, Giger W. 1987. Simultaneous determination of linear alkylbenzenesulfonates, alkylphenol polyethoxylates, and nonylphenol by high-performance liquid chromatography. Anal Chem 59: 1709-1715
Maruyama K, Yuan, M., and Otsuki, A. 2000. Seasonal Changes in Ethylene Oxide Chain Length of Poly(oxyethylene)alkylphenyl Ether Nonionic Surfactants in Three Main Rivers in Tokyo. Environ. Sci. Technol. 34: 343-348
Montgomery-Brown J, Drewes JE, Fox P, Reinhard M. 2003a. Behavior of alkylphenol polyethoxylate metabolites during soil aquifer treatment. Water Res 37: 3672-3681
Montgomery-Brown J, Reinhard M. 2003b. Occurrence and behavior of alkylphenol polyethoxylates in the environment. ENVIRON ENG SCI 20: 471-486
Pearce BA, Heydeman MT. 1980. Metabolism of di(ethylene glycol)[2-(2'-hydroxyethoxy)ethanol] and other short poly(ethylene glycol)s by gram-negative bacteria. J. Gen. Microbiol. 118: 21-27
Potter TL, Simmons K, Wu JN, Sanchezolvera M, Kostecki P. 1999. Static die-away of a nonylphenol ethoxylate surfactant in estuarine water samples. Environ . Sci. Technol. 33: 113-118
Ratti S, Curti B, Zanetti G, Galli E. 1985. Purification and characterization of glutamate synthase from Azospirillum brasilense. J Bacteriol 163: 724-729
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
Schink B, Janssen PH, Frings J. 1992. Microbial degradation of natural and of new synthetic polymers. FEMS Microbiol Rev 9: 311-316
Schroder HF. 2001. Tracing of surfactants in the biological wastewater treatment process and the identification of their metabolites by flow injection-mass spectrometry and liquid chromatography-mass spectrometry and -tandem mass spectrometry. J Chromatogr A 926: 127-150
Spengler P, Korner W, Metzger JW. 2001. Substances with estrogenic activity in effluents of sewage treatment plants in southwestern Germany. 1. Chemical analysis. Environ Toxicol Chem 20: 2133-2141
Stephanou E, Giger W. 1982. Persistent organic chemicals in sewage effluents. 2. Quantitative determinations of nonylphenols and nonylphenol ethoxylates by glass capillary gas chromatography. Environ. Sci. Technol. 16: 800-805
Sugimoto M, Tanabe M, Hataya M, Enokibara S, Duine JA, Kawai F. 2001. The first step in polyethylene glycol degradation by sphingomonads proceeds via a flavoprotein alcohol dehydrogenase containing flavin adenine dinucleotide. J Bacteriol 183: 6694-6698
Talmage SS. 1994. Environmental and human safety of major surfactants : alcohol ethoxylates and alkylphenol ethoxylates. Boca Raton. FL: Lewis Publishers
Tasaki Y, and Tamura, H. 2003. Characterization of dehydrogenase involved in biodegradation of octylphenol polyethoxylate in Pseudomonas putida. Unpublished
Thelu J, Medina L, Pelmont J. 1980. Oxidation of polyoxyethylene oligomers by an inducible enzyme from Pseudomonas P 400. FEMS Microbiol. Lett. 8: 187-190
Thiele B, Gunther K, J. SM. 1997a. Alkylphenol ethoxylates: Trace analysis and environmental behavior. Chem. Rev. 97: 3247-3272
Thiele B, Gunther K, Schwuger MJ. 1997b. Alkylphenol Ethoxylates: Trace Analysis and Environmental Behavior. Chem Rev 97: 3247-3272
Vanoni MA, Curti B. 1999. Glutamate synthase: a complex iron-sulfur flavoprotein. Cell Mol Life Sci 55: 617-638
Vanoni MA, Verzotti E, Zanetti G, Curti B. 1996. Properties of the recombinant beta subunit of glutamate synthase. Eur J Biochem 236: 937-946
White GF, Russell NJ, Tidswell EC. 1996. Bacterial scission of ether bonds. Microbiol Rev 60: 216-232
Yang Z. 1987. Synthesis of Triton X-100 used in liquid scintillation measurement. He Dianzixue Yu Tance Jishu 7: 312-314 |