Directed Evolution of Artificial Metalloenzymes: Genetic Optimization of the Catalytic Activity

Abstract

Artificial metalloenzymes (ArMs) based on the incorporation of a biotinylated metal cofactor within a streptavidin (Sav) combine attractive features of both enzymatic and homogeneous catalysis. To speed up their optimization, we present a directed evolution of an artificial transfer hydrogenase (ATHase) based on a stream-lined and optimized protocol for the design, overexpression and screening of Sav isoforms. Ten positions have been subjected to mutagenesis to yield two variants with improved catalytic activity and selectivity for the reduction of cyclic imines, along with greater stability in a biphasic medium.