1 research outputs found
Rapid Kinetic Characterization of Glycosyl Hydrolases Based on Oxime Derivatization and Nanostructure-Initiator Mass Spectrometry (NIMS)
Glycoside
hydrolases (GHs) are critical to cycling of plant biomass in the environment,
digestion of complex polysaccharides by the human gut microbiome,
and industrial activities such as deployment of cellulosic biofuels.
High-throughput sequencing methods show tremendous sequence diversity
among GHs, yet relatively few examples from the over 150,000 unique
domain arrangements containing GHs have been functionally characterized.
Here, we show how cell-free expression, bioconjugate chemistry, and
surface-based mass spectrometry can be used to study glycoside hydrolase
reactions with plant biomass. Detection of soluble products is achieved
by coupling a unique chemical probe to the reducing end of oligosaccharides
in a stable oxime linkage, while the use of <sup>13</sup>C-labeled
monosaccharide standards (xylose and glucose) allows quantitation
of the derivatized glycans. We apply this oxime-based nanostructure-initiator
mass spectrometry (NIMS) method to characterize the functional diversity
of GHs secreted by <i>Clostridium thermocellum</i>, a model
cellulolytic organism. New reaction specificities are identified,
and differences in rates and yields of individual enzymes are demonstrated
in reactions with biomass substrates. Numerical analyses of time series
data suggests that synergistic combinations of mono- and multifunctional
GHs can decrease the complexity of enzymes needed for the hydrolysis
of plant biomass during the production of biofuels