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 Km and Vmax values were 25.99 µM min−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 1 min. 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.
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Acknowledgements
This work was supported by grants from National Key Research and Development Project (SQ2018YFA090024), the Science and Technology Commission of Shanghai Municipality (17JC1403300), by the “Shuguang Program” (17SG09) supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission, and by grants from the Chinese National Science Foundation for Excellent Young Scholars (31422004).
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HZT outset and designed experiments. MD, ZLG and WW performed experiments. HZT and PX contributed reagents and materials. MD performed all the experiment and data analyses and wrote the manuscript. All authors discussed and revised the manuscript. All authors commented on the manuscript before submission. All authors read and approved the final manuscript.
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The Pseudomonas sp. strain SMT-1 has been deposited in the China Center for Type Culture Collection (CCTCC) under accession number AB2018208. The whole-genome shotgun sequencing data have been deposited at DDBJ/ENA/Gen Bank under accession number QJOV00000000. The version described in this article is the first version QJOV01000000.
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Desta, M., Zhang, L., Wang, W. et al. Molecular mechanisms and biochemical analysis of fluorene degradation by the Pseudomonas sp. SMT-1 strain. 3 Biotech 11, 416 (2021). https://doi.org/10.1007/s13205-021-02946-x
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DOI: https://doi.org/10.1007/s13205-021-02946-x