Genetic Optimization of the Catalytic Efficiency of Artificial Imine Reductases Based on Biotin-Streptavidin Technology

Artificial metalloenzymes enable the engineering of the reaction microenvironment of the active metal catalyst by modification of the surrounding host protein. We report herein the optimization of an artificial imine reductase (ATHase) based on biotin-streptavidin technology. By introduction of lipophilic amino acid residues around the active site, an 8-fold increase in catalytic efficiency compared with the wild type imine reductase was achieved. Whereas substrate inhibition was encountered for the free cofactor and wild type ATHase, two engineered systems exhibited classical Michaelis-Menten kinetics, even at substrate concentrations of 150 mM with measured rates up to 20 min(-1).