To design and optimize chemical reactors and bio reactors a profound understanding of kinetics and in particular suitable mechanistic rate models is needed. In chemical reaction engineering the methodology from Christiansen considering elementary kinetics assuming rate-determining steps has been successfully applied. It is hardly considered for enzymatic reactions. In this work, kinetics of formation of the prebiotics galacto-oligosaccharides (GOS) from lactose were investigated with 8-galactosidase using batch and dynamic experiments. The rates of the main and most relevant side reactions were quantified. Preliminary investigations were performed to determine the influence of pH, temperature, the amount of enzyme and the initial lactose concentration. To acquire mechanistic understanding instructive perturbation experiments were carried out dosing the substrate lactose and/or the products galactose and glucose either initially or dynamically during fermentation. Based on a postulated catalytic cycle, mechanistic kinetic models were developed considering hydrolysis of lactose, formation of GOS and inhibiting effects of the side products glucose and galactose. A sensitivity analysis was performed applying the subset selection method including model reduction based on singular value decomposition combined with rank revealing QR factorization. The parameterized mechanistic models allowed a good description of batch and dynamic experiments in a broad range of operating conditions. (C) 2017 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.