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Appl Environ Microbiol, January 1998, p. 153-158, Vol. 64, No. 1
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Morpholine Degradation Pathway of Mycobacterium aurum MO1: Direct Evidence of Intermediates by In Situ ¹H Nuclear Magnetic Resonance

B. Combourieu,¹ P. Besse,¹ M. Sancelme,¹ H. Veschambre,¹ A. M. Delort,^1,* P. Poupin,² and N. Truffaut²

Laboratoire de Synthèse, Electrosynthèse et Etude de Systèmes à Intérêt Biologique, UMR 6504 CNRS, Université Blaise Pascal, 63177 Aubière Cedex,¹ and Laboratoire de Génétique Microbienne, Université de Technologie de Compiègne, B.P. 649, 60206 Compiègne,² France

Received 23 June 1997/Accepted 31 August 1997

Resting Mycobacterium aurum MO1 cells were incubated with morpholine, a waste from the chemical industry. The kinetics of biodegradation was monitored by using in situ nuclear magnetic resonance (NMR). The incubation medium was directly analyzed by ¹H NMR. This technique allowed the unambiguous identification of two intermediates of the metabolic pathway involved in the biodegradation process, glycolate and 2-(2-aminoethoxy)acetate. The latter compound, which was not commercially available, was synthesized, in three steps, from 2-(2-aminoethoxy)ethanol. Quantitative analysis of the kinetics of degradation of morpholine was performed by integrating the signals of the different metabolites in ¹H-NMR spectra. Morpholine was degraded within 10 h. The intermediates increased during the first 10 h and finally disappeared after 20 h incubation. Assays of degradation were also carried out with glycolate and ethanolamine, hypothetical intermediates of the morpholine degradation pathway. They were degraded within 4 and 8 h, respectively. Until now, no tool for direct detection of intermediates or even morpholine has been available, consequently, only hypothetical pathways have been proposed. The approach described here gives both qualitative and quantitative information about the metabolic routes used in morpholine degradation by M. aurum MO1. It could be used to investigate many biodegradative processes.

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^* Corresponding author. Mailing address: Laboratoire de Synthèse, Electrosynthèse et Etude de Systèmes à Intérêt Biologique, UMR 6504 CNRS, Université Blaise Pascal, 63177 Aubière Cedex, France. Phone: 33 4 73 40 77 14. Fax: 33 4 73 40 77 17. E-mail: amdelort{at}chimtp.univ-bpclermont.fr.

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