Determination of glycoside hydrolase specificities during hydrolysis of plant cell walls using glycome profiling.
BACKGROUND
Glycoside hydrolases (GHs) are enzymes that hydrolyze polysaccharides into simple sugars. To better understand the specificity of enzyme hydrolysis within the complex matrix of polysaccharides found in the plant cell wall, we studied the reactions of individual enzymes using glycome profiling, where a comprehensive collection of cell wall glycan-directed monoclonal antibodies are used to detect polysaccharide epitopes remaining in the walls after enzyme treatment and quantitative nanostructure initiator mass spectrometry (oxime-NIMS) to determine soluble sugar products of their reactions.


RESULTS
Single, purified enzymes from the GH5_4, GH10, and GH11 families of glycoside hydrolases hydrolyzed hemicelluloses as evidenced by the loss of specific epitopes from the glycome profiles in enzyme-treated plant biomass. The glycome profiling data were further substantiated by oxime-NIMS, which identified hexose products from hydrolysis of cellulose, and pentose-only and mixed hexose-pentose products from the hydrolysis of hemicelluloses. The GH10 enzyme proved to be reactive with the broadest diversity of xylose-backbone polysaccharide epitopes, but was incapable of reacting with glucose-backbone polysaccharides. In contrast, the GH5 and GH11 enzymes studied here showed the ability to react with both glucose- and xylose-backbone polysaccharides.


CONCLUSIONS
The identification of enzyme specificity for a wide diversity of polysaccharide structures provided by glycome profiling, and the correlated identification of soluble oligosaccharide hydrolysis products provided by oxime-NIMS, offers a unique combination to understand the hydrolytic capabilities and constraints of individual enzymes as they interact with plant biomass.
US Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706 USA.|Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 USA.
Johnnie A
JA
Walker
US Department of Energy Bioenergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831 USA.|Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602 USA.
Sivakumar
S
Pattathil
US Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706 USA.|Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 USA.
Lai F
LF
Bergeman
US Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706 USA.|Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 USA.
Emily T
ET
Beebe
US Department of Energy Joint Bioenergy Institute, Emeryville, CA 94608 USA.|Sandia National Laboratories, Livermore, CA 94551 USA.
Kai
K
Deng
US Department of Energy Bioenergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831 USA.|Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602 USA.
Maryam
M
Mirzai
US Department of Energy Joint Bioenergy Institute, Emeryville, CA 94608 USA.|Lawrence Berkeley National Laboratory, Berkeley, CA 94720 USA.
Trent R
TR
Northen
US Department of Energy Bioenergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831 USA.|Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602 USA.
Michael G
MG
Hahn
US Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706 USA.|Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 USA.
Brian G
BG
Fox
Enzyme specificity; Glycome profiling; Glycoside hydrolase; Nanostructure-initiator mass spectrometry; Xylanase; Xyloglucanase