Abstract
Fusion enzymes are attractive tools for facilitating the assembly of biocatalytic cascades for chemical synthesis. This approach can offer great advantages for cooperative redox cascades that need the constant supply of a donor molecule. In this work, we have developed a self-sufficient bifunctional enzyme that can be coupled to transaminase-catalyzed reactions for the efficient recycling of the amino donor (L-alanine). By genetic fusion of an alanine dehydrogenase (AlaDH) and a formate dehydrogenase (FDH), a redox-complementary system was applied to recycle the amino donor and the cofactor (NADH), respectively. AlaDH and FDH were assembled in both combinations (FDH-AlaDH and AlaDH-FDH), with a 2.5-fold higher enzymatic activity of the latter system. Then, AlaDH-FDH was coupled to two different S-selective transaminases for the synthesis of vanillyl amine (10 mM) reaching up to 99 % conversion in 24 h in both cases. Finally, the multienzyme system was reused for at least 3 consecutive cycles when implemented in dialysis-assisted biotransformations.