Calorimetric evaluation of the activity and the mechanism of cellulases for the hydrolysis of cello-oligosaccharides accompanied by the mutarotation reaction of the hydrolyzed products

Isothermal titration calorimetry (ITC) combined with normal-phase HPLC has become one of the most widely applied methods for evaluating the kinetics of cellulase-catalyzed hydrolysis of cello-oligosaccharides, since it allows highly sensitive and precise measurement of the hydrolysis heat of single beta-1.4 glycosidic bonds. In this paper, we first extend the previously published analysis of the time course of the enzymatic reaction [N. Karim, S. Kidokoro, Thermochim. Acta 91-96 (2004) 412] in order to evaluate the hydrolysis heat during the enzyme injection, including information of the reaction in the early stage of the enzyme reaction. We then further extend the method in order to evaluate the heat effect accompanying the mutarotation of the newly produced reducing end of the products, which cannot be neglected in neutral pH. By using this method, the activities of two endoglucanases-an inverting-type enzyme and a retaining-type enzyme in the mechanism of the enzymatic reactions-were successfully evaluated against cello-oligosaccharides at pH 4.0 and 7.0. While the observed heats at pH 4.0 were explained by a simple hydrolysis reaction without considering the mutarotation effect, the ITC data at pH 7.0 clearly showed the presence of post-hydrolysis and were explained very well by the new model considering the heat of the mutarotation reaction of the products. The exothermic heat or the endothermic heat caused by the mutarotation reaction was obtained in the case of the inverting- or retaining-type cellulase, respectively. These results indicate that the calorimetric evaluation of the activity of endoglucanases can be done even in cases in which the mutarotation is not negligible, and that the sign of the mutarotation heat can be used to distinguish the anomeric type of the newly produced reducing end of the hydrolyzed products. (c) 2005 Elsevier B.V. All rights reserved.