title: Determination of the action modes of cellulases from hydrolytic profiles over a time course using fluorescence-assisted carbohydrate electrophoresis.
abstract: Fluorescence-assisted carbohydrate electrophoresis (FACE) is a sensitive and simple method for the separation of oligosaccharides. It relies on labeling the reducing ends of oligosaccharides with a fluorophore, followed by PAGE. Concentration changes of oligosaccharides following hydrolysis of a carbohydrate polymer could be quantitatively measured continuously over time using the FACE method. Based on the quantitative analysis, we suggested that FACE was a relatively high-throughput, repeatable, and suitable method for the analysis of the action modes of cellulases. On account of the time courses of their hydrolytic profiles, the apparent processivity was used to show the different action modes of cellulases. Cellulases could be easily differentiated as exoglucanases, β-glucosidases, or endoglucanases. Moreover, endoglucanases from the same glycoside hydrolases family had a variety of apparent processivity, indicating the different modes of action. Endoglucanases with the same binding capacities and hydrolytic activities had similar oligosaccharide profiles, which aided in their classification. The hydrolytic profile of Trichoderma reesei Cel12A, an endoglucanases from T. reesei, contained glucose, cellobiose, and cellotriose, which revealed that it may have a new glucosidase activity, corresponding to that of EC 3.2.1.74. A hydrolysate study of a T. reesei Cel12A-N20A mutant demonstrated that the FACE method was sufficiently sensitive to detect the influence of a single-site mutation on enzymatic activity.
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhang
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Zhang
authors.forename: Peipei
authors.initials: P
authors.lastname: Wang
authors.forename: Dandan
authors.initials: D
authors.lastname: Li
authors.forename: Guanjun
authors.initials: G
authors.lastname: Chen
authors.forename: Peiji
authors.initials: P
authors.lastname: Gao
authors.forename: Lushan
authors.initials: L
authors.lastname: Wang
mesh_terms: D044602:Cellulases / Q000378:metabolism*; D004586:Electrophoresis / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry*; D005656:Fungal Proteins / Q000378:metabolism; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D005656:Fungal Proteins; D009844:Oligosaccharides; D044602:Cellulases
keywords: Action mode; Cellulase; Fluorescence-assisted carbohydrate electrophoresis; Quantitative analysis