title: Continuous colorimetric screening assays for the detection of specific L- or D-α-amino acid transaminases in enzyme libraries.
abstract: In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of L- or D-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays' quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since L-CSA and D-CSA allowed the exclusive detection of L-TA and D-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable L- or D-homophenylalanine.
authors.affiliation: Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, Clermont-Ferrand, F 63000, France.|CNRS, UMR6296, ICCF, BP 80026, Aubière, F-63177, France.
authors.forename: Egon
authors.initials: E
authors.lastname: Heuson
authors.affiliation: CEA, DSV, IG, Genoscope, 2 rue Gaston Crémieux, Evry, 91057, France.|CNRS-UMR8030, 2 rue Gaston Crémieux, Evry, 91057, France.|Université d'Evry Val d'Essonne, Boulevard François Mitterrand, Evry, 91057, France.
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Petit
authors.affiliation: CEA, DSV, IG, Genoscope, 2 rue Gaston Crémieux, Evry, 91057, France.|CNRS-UMR8030, 2 rue Gaston Crémieux, Evry, 91057, France.|Université d'Evry Val d'Essonne, Boulevard François Mitterrand, Evry, 91057, France.
authors.forename: Adrien
authors.initials: A
authors.lastname: Debard
authors.affiliation: Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, Clermont-Ferrand, F 63000, France.|CNRS, UMR6296, ICCF, BP 80026, Aubière, F-63177, France.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Job
authors.affiliation: Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, Clermont-Ferrand, F 63000, France.|CNRS, UMR6296, ICCF, BP 80026, Aubière, F-63177, France.
authors.forename: Franck
authors.initials: F
authors.lastname: Charmantray
authors.affiliation: CEA, DSV, IG, Genoscope, 2 rue Gaston Crémieux, Evry, 91057, France. vberard@genoscope.cns.fr.|CNRS-UMR8030, 2 rue Gaston Crémieux, Evry, 91057, France. vberard@genoscope.cns.fr.|Université d'Evry Val d'Essonne, Boulevard François Mitterrand, Evry, 91057, France. vberard@genoscope.cns.fr.
authors.forename: Véronique
authors.initials: V
authors.lastname: de Berardinis
authors.affiliation: Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, Clermont-Ferrand, F 63000, France. thierry.gefflaut@univ-bpclermont.fr.|CNRS, UMR6296, ICCF, BP 80026, Aubière, F-63177, France. thierry.gefflaut@univ-bpclermont.fr.
authors.forename: Thierry
authors.initials: T
authors.lastname: Gefflaut
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D003124:Colorimetry / Q000379:methods*; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D057166:High-Throughput Screening Assays / Q000379:methods*; D013447:Sulfites / Q000378:metabolism; D000637:Transaminases / Q000032:analysis*
chemical_list: D000596:Amino Acids; D013447:Sulfites; D000637:Transaminases; D003545:Cysteine; C013461:cysteine sulfinic acid
keywords: Aminotransferase; Colorimetric assay; Cysteine sulfinic acid; Enzyme library; High-throughput screening; Transaminase