Sucrose phosphorylase (SPase) from Leuconostoc mesenteroides exhibited activity towards eight ketohexoses, which behaved as D-glucosyl acceptors, and alpha-D-glucose-l-phosphate (GIP), which behaved as a donor. All eight of these ketohexoses were subsequently transformed into the corresponding D-glucosyl-ketohexoses. Of the eight ketohexoses evaluated in the current study, D-allulose behaved as the best substrate for SPase, and the resulting D-glucosyl-D-alluloside product was found to be a non-reducing sugar with a specific optical rotation of [alpha](D)(20) + 74.36 degrees. D-Glucosyl-D-alluloside was identified as alpha-D-glucopyranosyl-(1 -> 2)-beta-D-allulofuranoside by NMR analysis. D-Glucosyl-D-alluloside exhibited an inhibitory activity towards an invertase from yeast with a K-m value of 50 mM, where it behaved as a competitive inhibitor with a K-i value of 9.2 mM. D-Glucosyl-D-alluloside was also successfully produced from sucrose using SPase and D-tagatose 3-epimerase. This process also allowed for the production of G1P from sucrose and D-allulose from D-fructose, which suggested that this method could be used to prepare D-glucosyl-D-alluloside without the need for expensive reagents such as G1P and D-allulose. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.