Among members of the glycoside hydrolase (GH) family, sucrose isomerase (SIase) and oligo-1,6glucosidase (O16G) are evolutionarily closely related even though their activities show different specificities. A gene (Avin_ 08330) encoding a putative SIase (AZOG: Azotobacter glucocosidase) from the nitrogen-fixing bacterium Azotobacter vinelandii is a type of pseudo-SIase harboring the " RLDRD" motif, a SIase-specific region in 329e333. However, neither sucrose isomerization nor hydrolysis activities were observed in recombinant AZOG (rAZOG). The rAZOG showed similar substrate specificity to Bacillus O16G as it catalyzes the hydrolysis of isomaltulose and isomaltose, which contain alpha-1,6-glycosidic linkages. Interestingly, rAZOG could generate isomaltose from the small substrate methyl-alpha-glucoside (MaG) via intermolecular transglycosylation. In addition, sucrose isomers isomaltulose and trehalulose were produced when 250 mM fructose was added to the MaG reaction mixture. The conserved regions I and II of AZOG are shared with many O16Gs, while regions III and IV are very similar to those of SIases. Strikingly, a shuffled AZOG, in which the N-terminal region of SIase containing conserved regions I and II was exchanged with the original enzyme, exhibited a production of sucrose isomers. This study demonstrates an evolutionary relationship between SIase and O16G and suggests some of the main regions that determine the specificity of SIase and O16G. (C) 2016 Elsevier Inc. All rights reserved.