Design of a novel redox biocatalyst to specification (363.3)

Recently, we obtained the first amine dehydrogenases, termed L-AmDH and F-AmDH, from leucine DH [1] and phenylalanine DH [2], respectively, allowing direct reductive amination of ketones to chiral amines. Starting from a scaffold devoid of such activity, a robust AmDH has been evolved with a single two-site library allowing for the direct production of (R)-1-(4-fluorophenyl)-propyl-2-amine from para-fluoro phenyl acetone with a kcat value of 6.85 s-1 at 25oC. Only (R)-amine was formed (> 99.8% e.e.), showing that perfect enantioselectivity of the template was conserved. Successful domain shuffling created a chimeric amine DH with enhanced substrate specificity that allows to accommodate sterically very demanding side chains, such as adamantly groups. We will elucidate the impact of restricted libraries in the evolution towards higher activity and broader specificity. Many interesting substrates feature low solubility in aqueous solution. We describe how utilizing a two-phase system can also lead to pinpointing broadened substrate specificity, as substrate candidates that were previously missed, can register as hits in the search.