Enhancement of the Substrate Scope of Transketolase

To enhance the activity of transketolase towards nonphosphorylated substrates and enlarge the scope of its substrates, notably to long polyol aldehyde acceptors ( D ‐ribose or D ‐glucose), a rational design‐supported evolution strategy was applied. By using docking experiments, an in silico library, and iterative mutagenesis, libraries of single‐ and double‐point mutants were designed and generated. A double‐screening approach was implemented, coupling a preselection activity assay (HPLC method) and a selective assay (GC method) to find the best enzymes. Several mutants (R526N, R526Q, R526Q/S525T, R526K/S525T) showed improved activities towards nonphosphorylated substrates as the coupled products of lithium hydroxypyruvate (HPA) with glycolaldehyde (GO), D ‐ribose or D ‐glucose. These mutated enzymes were further characterised. They were shown to be up to four times more active than the wild‐type (mutant R526Q/S525T) for nonphosphorylated substrates LiHPA/GO ( V m / K m for LiHPA = 92.4 instead of 28.8×10 −3 min −1 for the wild‐type) and 2.6 times more active for substrates LiHPA/rib.