Transketolase (TK) from various origins (including Escherichia coli and yeast) has been described to be fully enantiomer specific for (2R)-hydroxyaldehyde substrates. A thermostable TK from Geobacillus stearothermophilus (TKgst) was found to display a minor reactivity for (2S)-hydroxylated aldehydes. To improve this activity by directed protein evolution, we have built a library of TKgst variants by site saturation mutagenesis on two key positions L382 and D470. The best TKgst double mutant L382D/D470S shows up to 4- and 5-fold higher activities towards L-lactaldehyde and L-glyceraldehyde as acceptor substrates, respectively. Preparative utility of this mutant was demonstrated by the one-step synthesis of valuable L-ribulose and its 5-deoxy analogue with the L-erythro (3S,4S) configuration, which were previously inaccessible by using common TK sources.
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- α-hydroxylated aldehydes