Triple mutant K66Q/S149G/N262C (TM_pheDH) of Rhodococcus phenylalanine dehydrogenase (pheDH) was engineered by directed evolution as the first enzyme for the highly enantioselective reductive amination of phenylacetone 1 and 4-phenyl-2-butanone 3, giving (R)amphetamine 2 and (R)-1-methyl-3-phenylpropylamine 4 in >98% ee, respectively. The new amine dehydrogenase TM_pheDH with special substrate specificity is a valuable addition to the amine dehydrogenase family with very limited number, for asymmetric reductive amination of ketone, an important reaction in sustainable pharmaceutical manufacturing. Molecular docking provided insight into the role of key mutations of pheDH, being useful for engineering new amine dehydrogenases with higher activity and unique substrate scope.

Directed evolution of P450pyr created I83M/I82T mutant for highly regioselective terminal hydroxylation of n-butanol to 1,4-butanediol, representing the first achievement of this hydroxylation reaction by chemical or enzymatic methods and an unique example of evolving a hydroxylase to switch the substrate acceptance from a hydrophobic to hydrophilic compound.