Steviol glycosides from the leaves of the plant Stevia rebaudiana are high-potency natural sweeteners but suffer from a lingering bitterness. The Lactobacillus reuteri 180 wild-type glucansucrase Gtf180-Delta N, and in particular its Q1140E-mutant, efficiently alpha-glucosylated rebaudioside A (RebA), using sucrose as donor substrate. Structural analysis of the products by MALDI-TOF mass spectrometry, methylation analysis and NMR spectroscopy showed that both enzymes exclusively glucosylate the Glc(beta 1 -> C-19 residue of RebA, with the initial formation of an (alpha 1 -> 6) linkage. Docking of RebA in the active site of the enzyme revealed that only the steviol C-19 beta-D-glucosyl moiety is available for glucosylation. Response surface methodology was applied to optimize the Gtf180-Delta N-Q1140E-catalyzed a-glucosylation of RebA, resulting in a highly productive process with a RebA conversion of 95% and a production of 115 g/L alpha-glucosylated products within 3 h. Development of a fed-batch reaction allowed further suppression of alpha-glucan synthesis which improved the product yield to 270 g/L. Sensory analysis by a trained panel revealed that glucosylated RebA products show a significant reduction in bitterness, resulting in a superior taste profile compared to RebA. The Gtf180-Delta N-Q1140E glucansucrase mutant enzyme thus is an efficient biocatalyst for generating a-glucosylated RebA variants with improved edulcorant/organoleptic properties.