New Glycosidase Substrates for Droplet-Based Microfluidic
Screening
- Publication date
- Publisher
Abstract
Droplet-based microfluidics is a
powerful technique allowing ultra-high-throughput
screening of large libraries of enzymes or microorganisms for the
selection of the most efficient variants. Most applications in droplet
microfluidic screening systems use fluorogenic substrates to measure
enzymatic activities with fluorescence readout. It is important, however,
that there is little or no fluorophore exchange between droplets,
a condition not met with most commonly employed substrates. Here we
report the synthesis of fluorogenic substrates for glycosidases based
on a sulfonated 7-hydroxycoumarin scaffold. We found that the presence
of the sulfonate group effectively prevents leakage of the coumarin
from droplets, no exchange of the sulfonated coumarins being detected
over 24 h at 30 °C. The fluorescence properties of these substrates
were characterized over a wide pH range, and their specificity was
studied on a panel of relevant glycosidases (cellulases and xylanases)
in microtiter plates. Finally, the β-d-cellobioside-6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate
substrate was used to assay cellobiohydrolase activity on model bacterial
strains (<i>Escherichia coli</i> and <i>Bacillus subtilis</i>) in a droplet-based microfluidic format. These new substrates can
be used to assay glycosidase activities in a wide pH range (4–11)
and with incubation times of up to 24 h in droplet-based microfluidic
systems