Utilization of naproxen by Amycolatopsis sp. Poz 14 and detection of the enzymes involved in the degradation metabolic pathway

Abstract

The pollution of aquatic environments by drugs is a problem for which scarce research has been conducted in regards of their removal. Amycolatopsis sp. Poz 14 presents the ability to biotransformation naphthalene at high efficiency, therefore, in this work this bacterium was proposed as an assimilator of naproxen and carbamazepine. Growth curves at different concentrations of naproxen and carbamazepine showed that Amycolatopsis sp. Poz 14 is able to utilize these drugs at a concentration of 50 mg L−1 as a source of carbon and energy. At higher concentrations, the bacterial growth was inhibited. The transformation kinetics of naproxen showed the total elimination of the compound in 18 days, but carbamazepine was only eliminated in 19.9%. The supplementation with cometabolites such as yeast extract and naphthalene (structure similar to naproxen) at 50 mg L−1, showed that the yeast extract shortened the naproxen elimination to 6 days and reached a higher global consumption rate compared to the naphthalene cometabolite. The biotransformation of carbamazepine was not improved by the addition of cometabolites. The partial sequencing of the genome of Amycolatopsis sp. Poz 14 detected genes encoding putative enzymes for the degradation of cyclic aromatic compounds and the activities of aromatic monooxygenase, catechol 1,2-dioxygenase and gentisate 1,2-dioxygenase exhibited their involving in the naproxen biodegradation. The HPLC–MS analysis detected the 5-methoxysalicylic acid at the end of the biotransformation kinetics. This work demonstrates that Amycolatopsis sp. Poz 14 utilizes naproxen and transforms it to 5-methoxysalicylic acid which is the initial compound for the catechol and gentisic acid metabolic pathway.

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Acknowledgements

Chromatographic analysis performed with the equipment of “Laboratorio Nacional para Servicios Especializados de Investigación, Desarrollo e Innovación (I + D + i) para Farmoquímicos y Biotecnológicos”, LANSEIDI-FarBiotec-CONACyT, which is part of Unidad de Desarrollo e Investigación en Bioprocesos (UDIBI)-IPN” are gratefully acknowledged. BMAS thanks the support of the Consejo Nacional de Ciencia y Tecnología (CONACyT) for the scholarship grant for master’s degree, also the authors wish to acknowledge the financial support provided by the Instituto Politécnico Nacional (IPN) México Grant SIP20195543. Finally, CGB, JACM and JJR appreciate the COFAA and EDI, IPN fellowships, and support from the SNI-CONACyT.

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Supplementary material 1—Growth curve of Amycolatopsis sp. Poz 14 with cometabolites in mineral medium supplemented with naproxen (a) and carbamazepine (b). Both cometabolites were added at 50 mg L-1 (TIF 99,622 kb)

Supplementary material 2—Kinetics of biotransformation of carbamazepine by Amycolatopsis sp. Poz 14 in mineral medium with the addition of naphthalene or yeast extract (YE) at 50 mg L-1. Determination of the kinetics of carbamazepine and growth by the consortium was carried out at an initial concentration of 50 mg L-1 and evaluated every 3 days until 18 days at 30ºC and 100 rpm in cometabolic culture using 50 mg L-1 of naphthalene (a) or YE (b). Data presented in the graph are the mean and standard error of 3 independent assays. (TIF 99,730 kb)

Supplementary material 3—Absence of naproxen at the end of the degradation kinetics by Amycolatopsis sp. Poz 14. Initial kinetic (down) and final kinetic (up) total ion counting (TIC) chromatograms of naproxen, obtained by MS, were compared. It was observed that naproxen amounts were totally degraded. (TIF 2,145 kb)

Supplementary material 4—Retention time of 5-methoxysalicylic acid during the degradation kinetic of naproxen by Amycolatopsis sp. Poz 14. The presence of 5-methoxysalicylic acid was corroborated by extracting its characteristic m/z signal from total m/z acquired data. 214.08, 273.17 and 357.17 m/z signals were extracted from total m/z signal acquired from the MS analysis of initial (down) and final (up) cell culture degradation of naproxen. Analyzed signals were assigned to one main peak at 2.79 min (TIF 1,567 kb)

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Alanis-Sánchez, B.M., Pérez-Tapia, S.M., Vázquez-Leyva, S. et al. Utilization of naproxen by Amycolatopsis sp. Poz 14 and detection of the enzymes involved in the degradation metabolic pathway. World J Microbiol Biotechnol 35, 186 (2019). https://doi.org/10.1007/s11274-019-2764-0

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Keywords

  • Naproxen
  • Carbamazepine
  • Amycolatopsis
  • 5-Methoxysalicylic acid
  • Catechol
  • Gentisic acid