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  • ACET sale br Materials and methods br Results

    2020-02-11


    Materials and methods
    Results and discussion
    Concluding remarks Enzymatic production of bioactive 9-cis-11-trans-conjugated linoleic ACET sale is part of a detoxification mechanism against linoleic acid which is a toxic and stress factor for many lactic bacteria such as Lactobacillus plantarum. The complete process of detoxification metabolism has been denominated as biohydrogenation of polyunsaturated fatty acids and consists of a very intricate group of reactions catalyzed by many proteins which have been partially studied and characterized at the present; however, the participation of other proteins catalyzing equivalent or similar reactions is not omitted due to the complexity of the global process and the fact that it has been demonstrated that many enzymes display multifunctional activities such as the binding of fatty acids. In this work we demonstrated that multifunctional-anchorless-surface α-enolase isolated from L. plantarum cells, is involved in the formation of bioactive 9-cis-11-trans-CLA through a concerted dehydration and isomerization of 10-hydroxy-12-cis-octadecenoic acid, additionally to its central role in glycolytic metabolism, thus proving for the first time that this protein is a side participant in the biohydrogenation process and plays a role in cell detoxification from polyunsaturated fatty acids such as linoleic acid, along with the linoleate isomerase complex. Biochemical optimization studies of the isolated enzyme revealed that the addition of MgCl2, glycerol, n-dodecyl-β-d-maltoside and acetonitrile resulted in maximum production of 9-cis-11-trans-CLA and maximum stability of the enolase when catalyzing the dehydration and isomerization reaction. This ACET sale results are relevant given that bioactive isomer 9-cis-11-trans-CLA possess many beneficial effects for humans and is currently produced by means of chemical synthesis. This work provides information about production of bioactive conjugated linoleic acid enzymatically synthesized in a concerted way from previously formed 10-hydroxy-12-cis-octadecenoic acid. Furthermore, a three-dimensional arrangement of the protein was calculated through homology modeling and molecular docking analysis were carried out with the substrate and the product in order to identify that the binding sites of these molecules are two hydrophobic superficial pockets located at opposite ends of the protein but connected through a channel where the catalysis of dehydration and isomerization of 10-hydroxy-12-cis-octadecenoic acid might occur. It is worth mentioning, that the binding sites of fatty acids is not in conflict with the glycolytic site since the latter is located at the upper side of the protein.
    Acknowledgements This work was financially supported by the Programa de Apoyos para Proyectos de Investigación e Innovación Tecnológica (PAPIIT) in Mexico (funds IN207013). A special thanks to Programa de Maestría y Doctorado en Ciencias Químicas, UNAM and Consejo Nacional de Ciencia y Tecnología (CONACyT) in Mexico for the fellowship CVU/Reg: 271399/223477. The authors wish to thank Carmen Márquez and Lucero Ríos from Instituto de Química, UNAM for their technical support in the analysis of fatty acids.
    Introduction Enolase is mainly responsible for catalyzing the interconversion of 2-phosphoglycerate (2-PGA) and phosphoenol pyruvate (PEP) in glycolytic pathway. The enzyme has also several pathogenic features such as plasminogen and fibronectin binding activities (Avilán et al., 2011, Bao et al., 2014, Toledo et al., 2012). Despite the non-glycolytic functions of enolase has not been clarified completely yet (Paludo et al., 2015), the enzyme has sophisticated roles such as being part of protein clusters related to transcription, development, growth, aging, death, apoptosis in cells and they have been named as “moonlighting” functions (Avilán et al., 2011, Paludo et al., 2015).