In Vitro Studies on the Initial Reactions of Anaerobic Ethylbenzene Mineralization

Anaerobic mineralization of ethylbenzene by the denitrifying bacterium Azoarcus sp. strain EB1 was recently shown to be initiated by dehydrogenation of ethylbenzene to 1-phenylethanol. 1-Phenylethanol is converted to benzoate (benzoyl coenzyme A) via acetophenone as transient intermediate. We developed in vitro assays to examine ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities in cell extracts of this strain. With p-benzoquinone as the electron acceptor, cell extracts of Azoarcus sp. strain EB1 catalyzed ethylbenzene oxidation at a specific rate of 10 nmol min(−1) [mg of protein](−1) and an apparent K(m) for ethylbenzene of approximately 60 μM. The membrane-associated ethylbenzene dehydrogenase activity was found to oxidize 4-fluoroethylbenzene and propylbenzene but was unable to transform 4-chloro-ethylbenzene, the ethyltoluenes, and styrene. Enzymatic ethylbenzene oxidation was stereospecific, with (S)-(−)-1-phenylethanol being the only enantiomer detected by chiral high-pressure liquid chromatography analysis. Moreover, cell extracts catalyzed the oxidation of (S)-(−)-1-phenylethanol but not of (R)-(+)-1-phenylethanol to acetophenone. When cell extracts were dialyzed, (S)-(−)-1-phenylethanol oxidation occurred only in the presence of NAD(+), suggesting that NAD(+) is the physiological electron acceptor of 1-phenylethanol dehydrogenase. Both ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities were present in Azoarcus sp. strain EB1 cells that were grown anaerobically on ethylbenzene, 1-phenylethanol, and acetophenone, but these activities were absent in benzoate-grown cells.