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ACS Chem Biol. 2014 Jul 18;9(7):1470-9. doi: 10.1021/cb5000289. Epub 2014 May 12.

Rapid kinetic characterization of glycosyl hydrolases based on oxime derivatization and nanostructure-initiator mass spectrometry (NIMS).

Author information

1
Joint BioEnergy Institute, Emeryville, California 94608, United States.

Abstract

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 (13)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 Clostridium thermocellum, 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.

PMID:
24819174
DOI:
10.1021/cb5000289
[Indexed for MEDLINE]

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