The present study evaluated the influence of water activity and lactose concentration on the synthesis of galactooligosaccharides (GOS), by means of a hyperthermophilic beta-glycosidase in an organic system. The production of GOS gradually grew as water activity increased in the reaction system; later, their synthesis decreased as water activity increased. The authors used the response surface methodology to study how different water activities and different concentrations of lactose influenced the synthesis of GOS and their length. In every case, the variable that proved to have the greatest effect on GOS synthesis was water activity. Maximum GOS3 synthesis was reached at a water activity interval of 0.44-0.57, with lactose concentrations of 0.06%-0.1%, while GOS4 and GOS5 maxima were reached at water activity intervals of 0.47-0.57 and 0.49-0.60, respectively. The research showed that higher water activity was required to synthesize GOS of greater length. Synthesis of GOS would then depend on the flexibility of the enzyme, which in turn would depend on water activity of the reaction system. This hypothesis was supported by experiments in which the reaction temperature was modified in order to change the flexibility of the enzyme, thus leading to longer GOS. (c) 2006 Wiley Periodicals, Inc.