Technische Universiteit Delft, Gebouw voor Scheikunde, Afdeling Biotechnologie, Julianalaan 136, Delft, The Netherlands.
Chembiochem. 2012 Sep 3;13(13):1921-31. doi: 10.1002/cbic.201200240. Epub 2012 Jul 23.
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.
为了提高转酮醇酶对非磷酸化底物的活性，并扩大其底物的范围，特别是针对长多元醇醛受体（D-核糖或 D-葡萄糖），应用了一种基于合理设计的进化策略。通过对接实验、计算机文库和迭代诱变，设计并生成了单点突变和双点突变文库。实施了双筛选方法，将预筛选活性测定（HPLC 方法）和选择性测定（GC 方法）相结合，以找到最佳酶。几种突变体（R526N、R526Q、R526Q/S525T、R526K/S525T）对非磷酸化底物的活性提高，表现为羟丙酮锂（HPA）与甘油醛（GO）、D-核糖或 D-葡萄糖的偶联产物。进一步对这些突变酶进行了表征。与野生型相比，它们对非磷酸化底物 LiHPA/GO 的活性提高了 4 倍（突变体 R526Q/S525T 的 V(m) /K(m) for LiHPA 为 92.4，而野生型为 28.8×10(-3) min(-1)），对 LiHPA/rib 的底物活性提高了 2.6 倍。
