Lactic acid bacteria are common in our everyday foods and they make an important contribution to healthy bacterial microflora of mucous membranes and gastrointestinal tract (probiotics). In the work presented here, we characterized three inulosucrase (and one levansucrase) enzymes from human associated lactobacilli. Only three inulosucrase enzymes had been characterized before the work reported in this thesis was initiated. Our studies provided the first time evidence that cells of Lactobacillus johnsonii NCC 533 and Lactobacillus gasseri DSM 20604 convert sucrose in inulin and inulin fructooligosaccharides (FOS) in situ, highlighting their probiotic potential. This study also indicated the occurrence of frequent genetic modifications in the ecological niches of these lactobacilli. The inulosucrase protein sequences that had become available were used in alignments to identify amino acids that are well conserved in inulosucrases but not in levansucrases. These residues were targets for site-directed mutagenesis studies aiming to determine their functional roles. The results of these mutagenesis studies provided a better fundamental understanding of differences between inulosucrase/levansucrase enzymes, and are important for the construction of inulosucrase mutants with higher transglycosylation specificity, higher catalytic rates, and different FOS/polymer size distribution. Finally, these studies resulted in elucidation of the first 3D structure of an inulosucrase protein (InuJ) of Lb. johnsonii NCC 533 (in collaboration with Tjaard Pijning and Bauke Dijkstra, Biophysical Chemistry). Based on this 3D structure, future work may succeed in elucidating the structural basis for the observed differences in product specificity (β(2-1) or β(2-6) linkages) of inulosucrase and levansucrase enzymes.