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

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”....

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Autores: Soriano Maldonado, Pablo, Andújar-Sánchez, Montserrat, Clemente- Jiménez, Josefa María, Rodríguez-Vico, Felipe, Las Heras-Vázquez, Francisco Javier, Martínez-Rodrígueza, Sergio
Tipo de recurso: artículo
Fecha de publicación:2015
País:España
Institución:Universidad Francisco de Vitoria
Repositorio:DDFV. Repositorio Institucional de la Universidad Francisco de Vitoria
Idioma:inglés
OAI Identifier:oai:ddfv.ufv.es:10641/3599
Acceso en línea:https://hdl.handle.net/10641/3599
Access Level:acceso abierto
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spelling Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49.Soriano Maldonado, PabloAndújar-Sánchez, MontserratClemente- Jiménez, Josefa MaríaRodríguez-Vico, FelipeLas Heras-Vázquez, Francisco JavierMartínez-Rodrígueza, SergioN-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.20152015-01-0120152015-01-01journal articlehttp://purl.org/coar/resource_type/c_6501SMURhttp://purl.org/coar/version/c_71e4c1898caa6e32info:eu-repo/semantics/articleapplication/pdfhttps://hdl.handle.net/10641/3599reponame:DDFV. Repositorio Institucional de la Universidad Francisco de Vitoriainstname:Universidad Francisco de VitoriaInglésengopen accesshttp://purl.org/coar/access_right/c_abf2Atribución-NoComercial-SinDerivadas 3.0 Españahttp://creativecommons.org/licenses/by-nc-nd/3.0/es/info:eu-repo/semantics/openAccessoai:ddfv.ufv.es:10641/35992026-06-11T12:44:57Z
dc.title.none.fl_str_mv Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49.
title Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49.
spellingShingle Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49.
Soriano Maldonado, Pablo
title_short Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49.
title_full Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49.
title_fullStr Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49.
title_full_unstemmed Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49.
title_sort Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49.
dc.creator.none.fl_str_mv Soriano Maldonado, Pablo
Andújar-Sánchez, Montserrat
Clemente- Jiménez, Josefa María
Rodríguez-Vico, Felipe
Las Heras-Vázquez, Francisco Javier
Martínez-Rodrígueza, Sergio
author Soriano Maldonado, Pablo
author_facet Soriano Maldonado, Pablo
Andújar-Sánchez, Montserrat
Clemente- Jiménez, Josefa María
Rodríguez-Vico, Felipe
Las Heras-Vázquez, Francisco Javier
Martínez-Rodrígueza, Sergio
author_role author
author2 Andújar-Sánchez, Montserrat
Clemente- Jiménez, Josefa María
Rodríguez-Vico, Felipe
Las Heras-Vázquez, Francisco Javier
Martínez-Rodrígueza, Sergio
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv
description 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.
publishDate 2015
dc.date.none.fl_str_mv 2015
2015-01-01
2015
2015-01-01
dc.type.none.fl_str_mv journal article
http://purl.org/coar/resource_type/c_6501
SMUR
http://purl.org/coar/version/c_71e4c1898caa6e32
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://hdl.handle.net/10641/3599
url https://hdl.handle.net/10641/3599
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
Atribución-NoComercial-SinDerivadas 3.0 España
http://creativecommons.org/licenses/by-nc-nd/3.0/es/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
Atribución-NoComercial-SinDerivadas 3.0 España
http://creativecommons.org/licenses/by-nc-nd/3.0/es/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:DDFV. Repositorio Institucional de la Universidad Francisco de Vitoria
instname:Universidad Francisco de Vitoria
instname_str Universidad Francisco de Vitoria
reponame_str DDFV. Repositorio Institucional de la Universidad Francisco de Vitoria
collection DDFV. Repositorio Institucional de la Universidad Francisco de Vitoria
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