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The use of dyed bacterial cellulose to monitor cellulase complex activity

Tiboni, Marcela, Grzybowski, Adelia, Passos, Maurício, Barison, Andersson, Lião, Luciano Morais, Campos, Francinete Ramos, Pontarolo, Roberto, Fontana, José Domingos
Cellulose 2012 v.19 no.6 pp. 1867-1877
cellulose, correlation, couplings, endo-1,4-beta-glucanase, enzymatic hydrolysis, enzyme activity, glucose, hydrolysis, monitoring, nuclear magnetic resonance spectroscopy, reducing sugars, sugar products, thin layer chromatography
Bacterial cellulose (BC) membranes covalently dyed with Remazol Brilliant Blue R (RBB) was used as an advantageous chromogenic substrate for the detection and measurement of the activity of cellulolytic enzymes. The covalent coupling was confirmed using nuclear magnetic resonance techniques. The BC membranes, both dyed and non-dyed, were saccharified with a 1:1 combination of (endoglucanases + cellobiohydrolases):β-glucosidase. Native BC required a lower enzyme loading for the release of 2 mg of reducing sugars. A higher loading was required for the RBB-BC, which, in turn, was more easily hydrolysed than both the native and the RBB-Whatman n.1 filter paper standard substrates. The enzymatic hydrolysis kinetics of RBB-BC confirmed the correlation between the release of reducing sugars and dyed products as indicated by the Pearson’s correlation coefficient (r = 0.9950). Thin layer chromatography was used to monitor glucose and traces of cellobiose as the main hydrolysis products from native BC, whereas RBB-cellobiose was markedly dominant over RBB-glucose in the case of RBB-BC hydrolysis.