title: Glycine oxidase based high-throughput solid-phase assay for substrate profiling and directed evolution of (R)- and (S)-selective amine transaminases.
abstract: Transaminases represent one of the most important enzymes of the biocatalytic toolbox for chiral amine synthesis as they allow asymmetric synthesis with quantitative yields and high enantioselectivity. In order to enable substrate profiling of transaminases for acceptance of different amines, a glycine oxidase and horseradish peroxidase coupled assay was developed. Transaminase activity is detected upon transfer of an amine group from an amino donor substrate to glyoxylate, generating glycine, which is subsequently oxidized by glycine oxidase, releasing hydrogen peroxide in turn. Horseradish peroxidase uses the hydrogen peroxide to produce benzoquinone, which forms a red quinone imine dye by a subsequent condensation reaction. As glycine does not carry a chiral center, both (R)- and (S)-selective transaminases accepting glyoxylate as amino acceptor are amenable to screening. The principle has been transferred to establish a high-throughput solid-phase assay which dramatically decreases the screening effort in directed evolution of transaminases, as only active variants are selected for further analysis.
authors.affiliation: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University , Felix Hausdorff-Str. 4, 17487 Greifswald, Germany.
authors.forename: Martin S
authors.initials: MS
authors.lastname: Weiß
authors.forename: Ioannis V
authors.initials: IV
authors.lastname: Pavlidis
authors.forename: Clare
authors.initials: C
authors.lastname: Vickers
authors.forename: Matthias
authors.initials: M
authors.lastname: Höhne
authors.forename: Uwe T
authors.initials: UT
authors.lastname: Bornscheuer
mesh_terms: D000588:Amines / Q000737:chemistry / Q000378:metabolism*; D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D019020:Directed Molecular Evolution; D056506:Geobacillus / Q000201:enzymology; D057166:High-Throughput Screening Assays; D015394:Molecular Structure; D012984:Software; D013237:Stereoisomerism; D013379:Substrate Specificity; D000637:Transaminases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000588:Amines; D000594:Amino Acid Oxidoreductases; C115925:glycine oxidase; D000637:Transaminases