Abstract
Taking advantage of the catalytic promiscuity of l-carbamoylase from Geobacillus stearothermophilus CECT43 (BsLcar) and N-succinyl-amino acid racemase from Geobacillus kaustophilus CECT4264 (GkNSAAR), we have evaluated the production of different optically pure l-α-amino acids starting from different racemic N-formyl- and N-carbamoyl-amino acids using a dynamic kinetic resolution approach. The enzymes were immobilized on two different solid supports, resulting in improved stability of the enzymes in terms of thermostability and storage when compared to the enzymes in solution. The bienzymatic system retained up to 80 % conversion efficiency after 20 weeks at 4 °C and up to 90 % after 1 week at 45 °C. The immobilization process also resulted in a great enhancement of the activity of BsLcar toward N-formyl-tryptophan, showing for the first time that substrate specificity of l-carbamoylases can be influenced by this approach. The system was effective for the biosynthesis of natural and unnatural l-amino acids (enantiomeric excess (e.e.) >99.5 %), such as l-methionine, l-alanine, l-tryptophan, l-homophenylalanine, l-aminobutyric acid, and l-norleucine, with a higher performance toward N-formyl-α-amino acid substrates. Biocatalyst reuse was studied, and after 10 reaction cycles, over 75 % activity remained.
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Notes
The pH of the solution was readjusted with NaOH after dissolution of the substrate.
The ratio of the immobilized BsLcar/GkNSAAR enzymes in terms of their activity was approximately 10:1.
Twenty-five milligrams of the A161 matrix was selected as the minimum amount to be used reproducibly.
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Acknowledgments
This work was supported by the Spanish Ministry of Education and Science, the European Social Fund (ESF), and the European Regional Development Fund (ERDF), through project BIO2011-27842, by the Andalusian Regional Council of Innovation, Science and Technology, through project TEP-4691, and by the European Cooperation in Science and Technology (COST) Action CM1303. P.S.-M. was supported by the University of Almería. S.M.-R. was supported by the Spanish Ministry of Science and Innovation. We thank Andy Taylor for critical discussion of the manuscript and Pedro Madrid-Romero for technical assistance. We also thank Carmen Hernández-Cervantes for assistance with the polarimetry measurements.
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Soriano-Maldonado, P., Las Heras-Vazquez, F.J., Clemente-Jimenez, J.M. et al. Enzymatic dynamic kinetic resolution of racemic N-formyl- and N-carbamoyl-amino acids using immobilized l-N-carbamoylase and N-succinyl-amino acid racemase. Appl Microbiol Biotechnol 99, 283–291 (2015). https://doi.org/10.1007/s00253-014-5880-7
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DOI: https://doi.org/10.1007/s00253-014-5880-7