Kinetic analysis and evaluation of galactose oxidase activation by hematin in the green oxidation of glycerol

Galactose oxidase (GAO) is the best-characterized member of a family of enzymes known as radical-copper oxidases, which use O2 as the final electron acceptor. GAO required an external oxidant to be fully activated and also an external scavenger of H2O2, the inhibiting product of the catalytic cycle. On the other hand, hematin (HEM) is a mimetic structure of heme peroxidases that also displays catalase-like activity. In this work, GAO was used in tandem with HEM towards glycerol (GLY) oxidation. Spectrometric and dissolved oxygen data are presented as evidence that the bi-catalytic system was active and stable. The optimized activity was 0.409 µmol h−1 UGAO−1, 5.2 times higher than with catalase. Optimum conditions were found at GAO 9.0 U/mL, GLY 1700 mM, HEM 108 µg/mL and a pH of 9.0. Kinetic modeling was used to propose a catalytic mechanism. The enzymatic constants KM and kb were obtained for GLY and galactose, the natural substrate of GAO. Moreover, the activation constant by HEM and the inhibition constant by H2O2 were estimated for GAO. Extrapolation of the model suggested a positive involvement of superoxide radicals in the activity of GAO, instead of triggering hematin inactivation.Fil: Parodi, Adrián Rodrigo. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; ArgentinaFil: Asteasuain, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Planta Piloto de Ingeniería Química. Universidad Nacional del Sur. Planta Piloto de Ingeniería Química; Argentina. Universidad Nacional del Sur. Departamento de Ingeniería Química; ArgentinaFil: Magario, Ivana. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; Argentin