A pH‐Based High‐Throughput Assay for Transketolase: Fingerprinting of Substrate Tolerance and Quantitative Kinetics |
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Authors: | Dong Yi Dr. Titu Devamani Juliane Abdoul‐Zabar Dr. Franck Charmantray Dr. Virgil Helaine Prof. Dr. Laurence Hecquet Prof. Dr. Wolf‐Dieter Fessner |
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Affiliation: | 1. Institut für Organische Chemie und Biochemie, Technische Universit?t Darmstadt, Petersenstrasse 22, 64287 Darmstadt (Germany);2. Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont‐Ferrand, UMR CNRS 6296, B.?P. 10448, 63177 Aubière (France) |
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Abstract: | A pH‐based high‐throughput assay method has been developed for the rapid and reliable measurement of transketolase (TK) activity. The method is based on the decarboxylation of lithium hydroxypyruvate (HPA) as a hydroxyacetyl donor with an aldehyde acceptor, using phenol red as the pH indicator. Upon release of carbon dioxide from HPA, the pH increase in the reaction mixture can be determined photometrically by the color change of the pH indicator. At low buffer concentration (2 mM triethanolamine, pH 7.5), the method is highly sensitive and allows continuous monitoring, for quantitative determination of the kinetic parameters. By using this method, the substrate specificities of the TK enzymes from Escherichia coli and Saccharomyces cerevisiae, as well as two active‐site‐modified variants of the E. coli TK (D469E, H26Y) were evaluated against a panel of substrate analogues; specific activities and kinetic constants could be rapidly determined. Substrate quality indicated by assay determination was substantiated with novel TK applications by using achiral 3‐hydroxypropanal and 4‐hydroxybutanal for preparative synthesis of chiral deoxyketose‐type products. Determination of ee for the latter could be performed by chiral GC analysis, with an unambiguous correlation of the absolute configuration from rotation data. This pH‐based assay method is broadly applicable and allows rapid, sensitive, and reliable screening of the substrate tolerance of known TK enzymes and variants obtained from directed evolution. |
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Keywords: | biocatalysis carboligation high‐throughput screening pH‐indicator assay substrate specificity transketolases |
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