| 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. |
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