The method of integrated kinetics and its applicability to the exo-glycosidase-catalyzed hydrolysis of p-nitrophenyl glycosides.

In the present work we suggest an efficient method, using the whole time course of the reaction, whereby parameters k cat , K m and product K I for the hydrolysis of a p- nitrophenyl glycoside by an exo -acting glycoside hydrolase can be estimated in a single experiment. Its applicability was demonstrated for three retaining exo -glycoside hydrolases, β-xylosidase from Aspergillus awamori, β-galactosidase from Penicillium sp. and α-galactosidase from Thermotoga maritima (TmGalA). During the analysis of the reaction course catalyzed by the TmGalA enzyme we had observed that a non-enzymatic process, mutarotation of the liberated α- d -galactose, affected the reaction significantly. [ABSTRACT FROM AUTHOR]