Precise and continuous observation of cellulase-catalyzed hydrolysis of cello-oligosaccharides using isothermal titration calorimetry

Heat is a characteristic of all chemical reactions, and isothermal titration calorimetry (ITC) provides a possible way to continuously detect the heat from catalytic reactions with high sensitivity and reproducibility. Cellulase, the enzyme of glycosyl hydrolase, catalyzes the cleavage of β-1,4 glycosidic bonds in cellulose. In this paper, ITC was applied to evaluate cellulase activity using cello-oligosaccharides as substrates. The hydrolysis heat of a single glycosidic bond of the substrate was successfully detected by combining ITC and normal-phase HPLC, and the time course of the enzymatic reaction was monitored continuously by ITC. The enzymatic parameters kcat and KM, obtained from calorimetric observables, clearly indicated that the reaction was well approximated by a simple Michaelis–Menten equation under the experimental conditions of this study. The normal-phase HPLC analysis was combined with the ITC approach to observe hydrolysis patterns and was found to be an effective and precise way to evaluate the activity of cellulase against cello-oligosaccharides.