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.
Here we have characterized the first transketolase (TK) from a thermophilic microorganism, Geobacillus stearothermophilus, which was expressed from a synthetic gene in Escherichia coli. The G. stearothermophilus TK (mTK gst ) retained 100% activity for one week at 50 8C and for 3 days at 65 8C, and has an optimum temperature range around 60-70 8C, which will be useful for preparative applications and for future biocatalyst development. The thermostability of the mTK gst allowed us to carry out an easy, one-step purification by heat shock treatment of crude cell extracts at 65 8C for 45 min, directly yielding 132 mg of pure mTK gst from 1 L of culture. The reaction rate of mTK gst with glycolaldehyde was 14 times higher at 70 8C than at 20 8C, and 4 times higher at 50 8C when compared to E. coli TK under identical conditions. When tested at 50 8C with other aldehydes as acceptors, mTK gst activity was approximately 3 times higher than those obtained at 20 8C. Applications of this new TK in biocatalysis were performed with hydroxypyruvate as donor and three different aldehydes as acceptors -glycolaldehyde, d-glyceraldehyde and butyraldehyde -from which the corresponding products l-erythrulose 1, dxylulose 2 and 1,3-dihydroxyhexan-2-one 3 were obtained, respectively. The optical rotations for products 1 and 2 indicate that the stereospecificity of mTK gst is identical to that of other TK sources, leading to a (3S) configuration. With the non-hydroxylated substrate, butanal, the ee value was 85% (3S), showing higher enantioselectivity than the E. coli TK (75% ee, 3S). Processes at elevated temperatures could offer opportunities to extend the applications of TK biocatalysis, by favoring hydrophobic aldehyde acceptor substrate solubility and tolerance towards non-conventional media.
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