dc.description.abstract | Alkylphenol polyethoxylates (APEOn), including octylphenol polyethoxylates and nonylphenol polyethoxylates, are nonionic surfactants and are used in numerous commercial and industrial products including detergents, dispersants and emulsifiers. Large quantities of surfactants are released into the environment and these influence the fate of organic compounds in soils. The degradation products of these compounds, APEOn (n = 0 ~ 3), have been demonstrated to act as environmental hormones with estrogen activity. Octylphenol polyethoxylates (OPEOn) are composed of an aromatic ring with a branched eight-carbon alkyl polyethoxylate chain (average n = 9.5 ethoxylate units) at the para position. Our previous results have indicated that among the thirty-seven isolates capable of degrading this compound, a Gram-negative rod Pseudomonas sp. stain 82.10.6, showed the fastest growth and highest oxygen consumption activity using OPEOn as a sole carbon source and a transformation substrate, respectively. In this study, three identification methods, BioLog breath printing, fatty acid methyl ester fingerprinting and 16S rDNA sequence analysis, plus a negative activity for gelatin hydrolysis, were used to identify Pseudomonas sp. stain 82.10.6 as a Pseudomonas nitroreducens and the isolate was designated stain TX1. The sequential cleavage of the ethoxylate unit of OPEOn was confirmed by oxygen consumption during whole-cell fermentation of stain TX1. Using oxygen consumption activity as an enzyme assay, a NADPH-dependent enzyme was purified from stain TX1 and shown to shorten the ethoxylate chain. The OPEOn-oxygen consumption enzyme was isolated to homogeneity by flow-through anion exchange chromatography followed by ammonium sulfate precipitation, hydrophobic-interaction chromatography, gel filtration chromatography and chromatofocusing. The purified enzyme showed a subunit and native molecular weights of 52.9 ± 1.0 kDa (by SDS-PAGE) and 98.1 ± 7.5 kDa (by gel filtration), respectively. The enzyme configuration was a2. The pI of the enzyme was shown to be 6.65 ± 0.06 and 7.27 by chromatofocusing and 2-D gel electrophoresis, respectively. The absorption spectrum of the purified enzyme shows maximal absorbance at 375 and 458 nm, and the profile showed it contains FAD in a molar ratio of 1.87, that is one mole of FAD per subunit. Several peptide sequences from the enzyme were analyzed by ESI-Q-TOF and by MALDI-Q-TOF and most closely matched the same dihydrolipoamide dehydrogenase from Pseudomonas fluorescens (with a highest coverage of 36% of the amino acid sequence of the enzyme). Using the peptide sequences, molecular weight, configuration and absorption spectrum, the purified enzyme was identified as a dihydrolipoamide dehydrogenase. This is the first report of the isolation and characterization of dihydrolipoamide dehydrogenase from P. nitroreducens. The known function of this enzyme in all species up to the present is as one of the components in 2-oxo acid dehydrogenase complex family, with the function of oxidizing dihydrolipoamide on acetyltransferase modification. However, a new and very unique function has been demonstrated by this study. It is a NADPH-dependent, oxygen consuming enzyme and when exogenous transition metal ions, like Fe or Mn, are added to the enzyme reaction, the oxygen consumption activity is enhanced. The enzyme catalyzed product of this enzyme has been shown to be shortened ethoxylate chains of OPEOn (n = 6 is dominant) by LC/MS analysis. The cleavage of the ethoxylate chain of OPEOn occurs by hydroxyl radical attack and this was previously proposed by other researchers as one possible ethoxylate chain degradation mechanism. The hydroxyl radical is one of the products from the Fenton reaction. Where Fenton reagents (reduced metals and H2O2) are produced, there is both a reduction of transition metal ions and consumption of oxygen (O2 → O2•- → HOO• → H2O2) by dihydrolipoamide dehydrogenase in the presence of excess NADPH. This novel discovery is the first time that such a function has been shown for a bacterial dihydrolipoamide dehydrogenase. | en_US |