The stereoselective three-enzyme cascade for the one-pot synthesis of (1S,2S)-1-phenylpropane-1,2-diol ((1S,2S)-1-PPD) from inexpensive starting substrates, benzaldehyde and acetaldehyde, was explored. By coupling stereoselective carboligation catalyzed by benzoylformate decarboxylase (BFD), L-selective reduction of a carbonyl group with alcohol dehydrogenase from Lactobacillus brevis (ADHLb) as well as the coenzyme regeneration by formate dehydrogenase (FDH), enantiomerically pure diastereoselective 1,2-diol was produced. Two different multi-enzyme system approaches were applied: the sequential two-step one-pot and the simultaneous one-pot cascade. All enzymes were kinetically characterized. The impact of acetaldehyde on the BFD and ADHLb stability was investigated. To overcome the kinetic limitation of acetaldehyde in the carboligation reaction and to reduce its influence on the enzyme stability, experiments were performed in two different excesses of acetaldehyde (100 and 300%). Due to the ADHLb deactivation by acetaldehyde, the simultaneous one-pot cascade proved not to be the first choice for the investigated three-enzyme system. In the sequential cascade with 300% acetaldehyde excess a 100% yield of vic 1,2-diol was reached.
Keywords: Alcohol dehydrogenase; Asymmetric reduction; Benzoylformate decarboxylase; Multi-enzyme cascade reaction; Stereoselectivity.