Kinetic model discrimination via step-by-step experimental and computational procedure in the enzymatic oxidation of D-glucose

The enzymatic oxidation of D-glucose to 2-keto-D-glucose (D-arabino-hexos-2 -ulose, D-glucosone) is of prospective industrial interest. Pyranose oxidas e (POx) from Peniphora gigantea is deactivated during the reaction. To deve lop a kinetic model including the main reaction and the enzyme inactivation , possible side-reactions of the non-stabilised enzyme with D-glucosone, hy drogen peroxide, and peroxide radicals were considered. A developed step-by -step combined experimental and computational procedure allowed to discrimi nate among alternative inactivation mechanisms and provides an increased mo del reliability. The most probable scheme is the enzyme inactivation by hyd roxyl radicals formed from produced H2O2 in the presence of Fe2+ ions. This (OH)-O-. reaction is supported by matrix assisted laser desorption ionisat ion-mass spectrometry (MALDI-MS) measurement. The estimated kinetic paramet er values for the main reaction are of the same order of magnitude as those reported in the literature. The identified model allows a satisfactory pro cess simulation and highlights measures to prevent the enzyme activity loss . (C) 2001 Elsevier Science B.V. All rights reserved.