title: Studies of enzymatic cleavage of cellulose using polysaccharide analysis by carbohydrate gel electrophoresis (PACE).
abstract: With the advent of fast genome analysis, many genes encoding novel putative cellulolytic enzymes are being identified in diverse bacterial and fungal genomes. The discovery of these genes calls for quick, robust, and reliable methods for qualitative and quantitative characterization of the enzymatic activities of the encoded proteins. Here, we describe the use of the polysaccharide analysis by carbohydrate gel electrophoresis (PACE) method, which was previously used, among other applications, to characterize various hemicellulose degrading enzymes; for structural elucidation of these carbohydrates; and for analysis of products resulting from enzymatic cleavage of cellulose. PACE relies on fluorescent labeling of mono-, oligo-, and polysaccharides at their reducing end and separation of the labeled carbohydrates by polyacrylamide gel electrophoresis. Labeling can be carried out before or after enzymatic digestion. PACE is very sensitive and allows analysis of both substrate specificities and kinetic properties of cellulolytic enzymes.
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Ondrej
authors.initials: O
authors.lastname: Kosik
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Bromley
authors.forename: Marta
authors.initials: M
authors.lastname: Busse-Wicher
authors.forename: Zhinong
authors.initials: Z
authors.lastname: Zhang
authors.forename: Paul
authors.initials: P
authors.lastname: Dupree
mesh_terms: D002241:Carbohydrates / Q000737:chemistry; D002480:Cellulase / Q000378:metabolism; D044602:Cellulases / Q000378:metabolism; D002482:Cellulose / Q000032:analysis / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D006868:Hydrolysis; D011134:Polysaccharides / Q000032:analysis* / Q000378:metabolism; D055327:Thermoascus / Q000201:enzymology
chemical_list: D002241:Carbohydrates; D005456:Fluorescent Dyes; D011134:Polysaccharides; C007916:hemicellulose; D002482:Cellulose; D044602:Cellulases; D002480:Cellulase