Engineered Enzymes that Retain and Regenerate their Cofactors Enable Continuous-Flow Biocatalysis

Biocatalysis is used for many chemical syntheses due to its high catalytic rates, specificities and operation under ambient conditions. Continuous-flow chemistry offers advantages to biocatalysis, avoiding process issues caused by substrate/product inhibition, equilibrium controlled limitations on yield and allosteric control. Modular continuous-flow biochemistry would also allow the flexible assembly of different complex multistep reactions. Here we tackle some technical challenges that currently prohibit the wide-spread use of continuous flow biocatalysis; cofactor immobilization and site-specific immobilization. We provide the first example of enzymes engineered to retain and recycle their cofactors, and the use of these enzymes in continuous production of chiral pharmaceutical intermediates.