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.

Determination of the modes of action and synergies of xylanases by analysis of xylooligosaccharide profiles over time using fluorescence‐assisted carbohydrate electrophoresis

“…C). The amount of xylobiose decreased gradually whereas that of xylose increased gradually, in a manner similar to that described previously for hydrolysis by 1,4‐β‐glucosidase . The change curves for xylobiose and xylose were well fitted by a one‐phase decay exponential equation with R 2 > 0.95 (Fig.…”

“…For each type of XOS, all coefficients of determination ( R 2 ) between loading volumes and gray values were > 0.99 (Fig. B), indicating that the gray values of the electrophoresis gels could reliably be used to quantify sample concentrations, as previously reported . The changes in concentration of the XOS were thus reflected by the changes in gray values, which could be quantitatively characterized by FACE.…”

“…Mixtures of reaction products (5 μL), obtained as described in Section , were labeled by fluorescence and determined by electrophoresis as previously described. To label the products, reaction hydrolysate or XOS (5 μL) was treated with 7‐amino‐1,3‐naphthalenedisulfonic acid monopotassium salt monohydrate in 15% acetic acid (0.2 M, 5 μL) and allowed to react in the dark for 1 h. NaCNBH 3 solution (1 M, 5 μL) was added to the mixture, which was then incubated at 40°C for 12 h. Labeled products (6 μL/well) were loaded for electrophoresis, a miniaturized vertical gel system using a mini‐PROTEAN 3 PowerPac Basic Power Supply (Bio‐Rad, Hercules, CA, USA) was utilized for electrophoresis, the components and concentrations for the polyacrylamide gels were as described , and a ChemiDoc™ MP imaging system (Bio‐Rad, Hercules, CA, USA) was used to scan the gels. The scanned images were stored in TIF format; the digital information of the bands was acquired mainly from the peaks extracted quantitatively using Quantity One software (Bio‐Rad).…”

“…F32 have been determined using thin‐layer chromatography , but the substituted XOS were not identified. We have successfully used fluorophore‐assisted carbohydrate electrophoresis (FACE) as a sensitive, simple, and quantitative method to study the mode of action of cellulases , and this prompted us to develop a method using FACE to study the modes of action and synergies of components of the more complex xylanase system.…”

Biochemical and structural characterization of a novel halotolerant cellulase from soil metagenome

“…The reaction was stopped by addition of DNS as described earlier. The hydrolyzed products released can be quantitatively estimated by FACE (Fluorescence-assisted carbohydrate electrophoresis)72. The activity on para -nitrophenyl-β-D-cellobioside and para -nitrophenyl-β-D-glucopyranoside was checked by incubating 50 μl of 10 mM substrate with 50 μl (0.2 μg) of diluted enzyme for 15 minutes at 58 °C.…”