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Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49

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Abstract

N-Succinyl-amino acid racemase (NSAAR), long referred to as N-acyl- or N-acetyl-amino acid racemase, is an enolase superfamily member whose biotechnological potential was discovered decades ago, due to its use in the industrial dynamic kinetic resolution methodology first known as “Acylase Process”. In previous works, an extended and enhanced substrate spectrum of the NSAAR from Geobacillus kaustophilus CECT4264 toward different N-substituted amino acids was reported. In this work, we describe the cloning, purification, and characterization of the NSAAR from Geobacillus stearothermophilus CECT49 (GstNSAAR). The enzyme has been extensively characterized, showing a higher preference toward N-formyl-amino acids than to N-acetyl-amino acids, thus confirming that the use of the former substrates is more appropriate for a biotechnological application of the enzyme. The enzyme showed an apparent thermal denaturation midpoint of 77.0 ± 0.1 °C and an apparent molecular mass of 184 ± 5 kDa, suggesting a tetrameric species. Optimal parameters for the enzyme activity were pH 8.0 and 55–65 °C, with Co2+ as the most effective cofactor. Mutagenesis and binding experiments confirmed K166, D191, E216, D241, and K265 as key residues in the activity of GstNSAAR, but not indispensable for substrate binding.

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Notes

  1. We will use the three names indistinctly during this work, to try to maintain the original nomenclature used in the corresponding papers.

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Acknowledgments

We thank Andy Taylor for critical discussion of the manuscript and Pedro Madrid-Romero for technical assistance. 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 the project BIO2011-27842, by the Andalusian Regional Council of Innovation, Science and Technology, through the 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.

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  1. Sergio Martínez-Rodríguez

    Present address: Department of Physical Chemistry, University of Granada, 18071, Granada, Spain

Authors and Affiliations

  1. Dpto. Química y Física, Universidad de Almería, Campus de Excelencia Internacional Agroalimentario, ceiA3, Edificio CITE I, Carretera de Sacramento s/n., 04120, La Cañada de San Urbano, Almería, Spain

    Pablo Soriano-Maldonado, Montserrat Andújar-Sánchez, Josefa María Clemente-Jiménez, Felipe Rodríguez-Vico, Francisco Javier Las Heras-Vázquez & Sergio Martínez-Rodríguez

  2. Centro de Investigación en Biotecnología Agroalimentaria, BITAL, Almería, Spain

    Pablo Soriano-Maldonado, Montserrat Andújar-Sánchez, Josefa María Clemente-Jiménez, Felipe Rodríguez-Vico, Francisco Javier Las Heras-Vázquez & Sergio Martínez-Rodríguez

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  1. Pablo Soriano-Maldonado
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Soriano-Maldonado, P., Andújar-Sánchez, M., Clemente-Jiménez, J.M. et al. Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49. Mol Biotechnol 57, 454–465 (2015). https://doi.org/10.1007/s12033-015-9839-4

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