High-throughput functional assessment of polysaccharide-active enzymes using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry as exemplified on plant cell wall polysaccharides.
Despite a wealth of sequence information on genes encoding carbohydrate-active enzymes (e.g., transferases, esterases, hydrolases), very few of these enzymes have been described in detail, particularly regarding substrate specificities. A facile and rapid method for the characterization of substrate specificities of polysaccharide-active enzymes that uses matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) has been developed. This method has been applied to characterize a xyloglucan fucosyltransferase and a pectin methyl-esterase. Reactions were performed in liquid phase, and aliquots of the reaction mixtures were spotted on a polyvinylidene fluoride (PVDF) membrane. Reaction products were precipitated onto the membrane and cleaned by treatment with an ethanol-water mixture. Subsequently, the reaction products were hydrolyzed by specific endoglycanases, and the resulting oligosaccharides were directly analyzed onto the PVDF membrane by MALDI-TOF MS. The new method is amenable to high-throughput analysis and, thus, constitutes an emerging avenue to rapidly fill the gap in our knowledge of the specificities of polysaccharide-active enzymes.
Leboeuf
Edouard
E
Department of Plant Physiology, Institute of Biochemistry and Biology, University of Potsdam, D-14476 Potsdam-Golm, Germany.
Immerzeel
Peter
P
Gibon
Yves
Y
Steup
Martin
M
Pauly
Markus
M
D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism*; D005647:Fucosyltransferases / Q000378:metabolism*; D010944:Plants / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013379:Substrate Specificity
D011134:Polysaccharides; D005647:Fucosyltransferases; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase