The aim of the study was to analyze galactooligosaccharide (GOS) synthesis catalyzed by beta-galactosidase from Aspergillus oryzae by applying experimental data to optimization and kinetic parameter estimation methods. The apparent kinetic parameters were determined in varying initial lactose concentrations by using six-step, 11-parameter GOS synthesis mechanism. The results revealed that GOS yield was mainly dependent on initial lactose concentration; on the other hand, all of the reaction parameters affected the required time to achieve maximum GOS yield (TMGY) significantly. The values of 32 degrees Brix, 36.8 degrees C, and 8.32 U/g lactose solution were determined as the optimum reaction experimental set which offers 26.73% GOS yield and 369.82 min TMGY. The increased enzyme-galactose complex dissociation ratio (k(3)/k(-3)) with decreasing initial lactose concentration resulted to low GOS productivity although GOS production/GOS dissociation rate ratio (k(4)/k(-4)) increased with decreasing initial lactose concentration. This result signified that hydrolysis characteristics dominated the reaction medium at low initial lactose concentration. The role of monosaccharide inhibition became important on GOS synthesis for increasing initial lactose concentration.