Monooxygenase-mediated degradation of TDTMA by Pseudomonas putida

LIFFOURRENA. A.S.; BOERIS, P.S.; SALVANO, M.A.; LUCCHESI, G.I.

Congreso; XL Reunión Anual. Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2004

Tetradecyltrimethylammonium (TDTMA) is a quaternary ammonium compound commonly released into the environment. P. putida tolerated high concentrations of TDTMA (up 50 mg l-1) and utilized it as a sole carbon and nitrogen source. Here, we report the complete degradation of TDTMA and the initial characterization of inducible monooxygenase activity implicated in the first degradation steps. This enzyme, which catalyzed the cleavage of N-R bound of TDTMA producing trimethylamine (TMA) and an alkyl residue, was measured with two methods: i) evaluating the formation of tetradecanoic acid, analyzing the methyl acid ester by GC MS, or ii) measuring the production of TMA by GC-MS in a reaction mixture containing 14 mM phosphate buffer pH 7.4, 0.5 mM NAD(P)H, 0.5 mM TDTMA and 0.01-0.35 mg protein of free-extract cells. The enzyme activity was O2 and NAD(P)H dependent and showed sigmoid velocity curves with respect to the substrate TDTMA. The napp (Hill coefficient) and [S]05 values were approximately 2,24 and 4,26.10-4 M, respectively. The addition of FAD (20-100 M) showed 20% enzyme activation and no significant effect was found with Al3+, Zn2+ , Mg2+ and Cu2+ ions or with metal-chelating agents as EDTA and o-phenanthroline. Taking into account that other bacteria can not grow with TDTMA as a sole C / N source, the presence of a monooxygenase activity revealed the potential of P. putida to metabolize quaternary ammonium compounds.