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Appl Environ Microbiol, January 1998, p. 159-165, Vol. 64, No. 1
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Degradation of Morpholine by an Environmental Mycobacterium Strain Involves a Cytochrome P-450

P. Poupin,¹ N. Truffaut,^1,* B. Combourieu,² P. Besse,² M. Sancelme,² H. Veschambre,² and A. M. Delort²

Laboratoire de Génétique Microbienne, Université de Technologie de Compiègne, 60206 Compiègne,¹ and Laboratoire de Synthèse, Electrosynthèse et Etude de Systèmes à Intérêt Biologique, UMR 6504 CNRS, Université Blaise Pascal, 63177 Aubière Cedex,² France

Received 23 June 1997/Accepted 31 August 1997

A Mycobacterium strain (RP1) was isolated from a contaminated activated sludge collected in a wastewater treatment unit of a chemical plant. It was capable of utilizing morpholine and other heterocyclic compounds, such as pyrrolidine and piperidine, as the sole source of carbon, nitrogen, and energy. The use of in situ ¹H nuclear magnetic resonance (¹H NMR) spectroscopy allowed the determination of two intermediates in the biodegradative pathway, 2-(2-aminoethoxy)acetate and glycolate. The inhibitory effects of metyrapone on the degradative abilities of strain RP1 indicated the involvement of a cytochrome P-450 in the biodegradation of morpholine. This observation was confirmed by spectrophotometric analysis and ¹H NMR. Reduced cell extracts from morpholine-grown cultures, but not succinate-grown cultures, gave rise to a carbon monoxide difference spectrum with a peak near 450 nm, which indicated the presence of a soluble cytochrome P-450. ¹H NMR allowed the direct analysis of the incubation medium containing metyrapone, a specific inhibitor of cytochrome P-450. The inhibition of morpholine degradation was dependent on the morpholine/metyrapone ratio. The heme-containing monooxygenase was also detected in pyrrolidine- and piperidine-grown cultures. The abilities of different compounds to support strain growth or the induction of a soluble cytochrome P-450 were assayed. The results suggest that this enzyme catalyzes the cleavage of the C---N bond of the morpholine ring.

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^* Corresponding author. Mailing address: Laboratoire de Génétique Microbienne, Université de Technologie de Compiègne, B.P. 529, 60206 Compiègne, France. Phone: 33 3 44 23 44 52. Fax: 33 3 44 20 48 13. E-mail: nicole.truffaut{at}utc.fr.

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