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Applied and Environmental Microbiology, September 2003, p. 5216-5221, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5216-5221.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Biotransformation of 2,4,6,8,10,12-Hexanitro-2,4,6,8,10,12-Hexaazaisowurtzitane (CL-20) by Denitrifying Pseudomonas sp. Strain FA1

Bharat Bhushan,¹ Louise Paquet,¹ Jim C. Spain,² and Jalal Hawari^1*

Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada,¹ U.S. Air Force Research Laboratory, Tyndall Air Force Base, Florida 32403²

Received 3 April 2003/ Accepted 18 June 2003

The microbial and enzymatic degradation of a new energetic compound, 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20), is not well understood. Fundamental knowledge about the mechanism of microbial degradation of CL-20 is essential to allow the prediction of its fate in the environment. In the present study, a CL-20-degrading denitrifying strain capable of utilizing CL-20 as the sole nitrogen source, Pseudomonas sp. strain FA1, was isolated from a garden soil. Studies with intact cells showed that aerobic conditions were required for bacterial growth and that anaerobic conditions enhanced CL-20 biotransformation. An enzyme(s) involved in the initial biotransformation of CL-20 was shown to be membrane associated and NADH dependent, and its expression was up-regulated about 2.2-fold in CL-20-induced cells. The rates of CL-20 biotransformation by the resting cells and the membrane-enzyme preparation were 3.2 ± 0.1 nmol h^-1 mg of cell biomass^-1 and 11.5 ± 0.4 nmol h^-1 mg of protein^-1, respectively, under anaerobic conditions. In the membrane-enzyme-catalyzed reactions, 2.3 nitrite ions (NO₂^-), 1.5 molecules of nitrous oxide (N₂O), and 1.7 molecules of formic acid (HCOOH) were produced per reacted CL-20 molecule. The membrane-enzyme preparation reduced nitrite to nitrous oxide under anaerobic conditions. A comparative study of native enzymes, deflavoenzymes, and a reconstituted enzyme(s) and their subsequent inhibition by diphenyliodonium revealed that biotransformation of CL-20 is catalyzed by a membrane-associated flavoenzyme. The latter catalyzed an oxygen-sensitive one-electron transfer reaction that caused initial N denitration of CL-20.

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* Corresponding author. Mailing address: Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada. Phone: (514) 496-6267. Fax: (514) 496-6265. E-mail: jalal.hawari{at}nrc.ca.

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Applied and Environmental Microbiology, September 2003, p. 5216-5221, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5216-5221.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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