Molecular mechanisms and biochemical analysis of fluorene degradation by the Pseudomonas sp. SMT-1 strain.

Abstract

Fluorene is a harmful organic toxicant extensively disseminated in the water and dry land ecosystem. Its toxicity and ubiquitous presence pose issues concerning its biodegradation. Characterization of the molecular mechanisms of fluorene degradation, detection of metabolites, and appraisal of its viability in toxicant removal by the SMT-1 Pseudomonas sp. strain are the main purposes of this study. In this work, the catabolic intermediates were identified from resting cell reactions of the SMT-1 strain as well as the involved catabolic pathway of fluorene. Based on liquid chromatography mass spectrometry analysis, the identified intermediates were 9-fluorenone; 3,4-dihydroxy-9-fluorenone; phthalate and protocatechuic acid. The specific primers were designed to amplify the fluorene-degrading 4921 dioxygenase gene segment from the SMT-1 Pseudomonas sp. strain. The 4921 dioxygenase gene was expressed, purified and characterized. The apparent K m and V max values were 25.99µMmin-1 and 0.77 U mg-1, respectively. The enzyme was most active at pH 7.5 and 25°C in Tris-HCl buffer and was identified by measuring the initial reaction velocity for 1min. Effect of metal salts on enzyme activity was accessed to see the impact on protein stability. Most of the analyzed metal salts inhibited enzyme activity to different degrees, and exhibited very low activity in the presence of FeCl3. Understanding the physiological, metabolic pathway and molecular mechanism of fluorene degradation is an important factor in increasing significant information of this biological process. This strain may serve as a potential candidate for further use in the bioremediation process to treat organic toxicant contaminated sites.