Here, we describe the successful engineering of a promiscuous glucose dehydrogenase from the thermophilic archaeon, Saccharolobus solfataricus, toward the thermostable and robust cofactor, carba-NADP+, using a photometric-based microfluidic screening system at elevated temperatures. We show that droplets encapsulating single enzyme variants with the right handling could sufficiently sustain long incubation time at elevated temperatures. Furthermore, we enabled an ultrahigh-throughput screening for this thermostable enzyme with a combined targeted/random mutagenesis approach. This allowed us to explore a wide sequence space of the glucose dehydrogenase and find two positions remote from the catalytic center. Finally, we identified glucose dehydrogenase variants with more than 10-fold increase in catalytic speed and efficiency toward the artificial nicotinamide cofactor and some that showed completely switched cofactor preference at high substrate concentrations.     «

Here, we describe the successful engineering of a promiscuous glucose dehydrogenase from the thermophilic archaeon, Saccharolobus solfataricus, toward the thermostable and robust cofactor, carba-NADP+, using a photometric-based microfluidic screening system at elevated temperatures. We show that droplets encapsulating single enzyme variants with the right handling could sufficiently sustain long incubation time at elevated temperatures. Furthermore, we enabled an ultrahigh-throughput screening f...     »