Carboxy-terminal region of a thermostable CITase from Thermoanaerobacter thermocopriae has the ability to produce long isomalto-oligosaccharides.

Isomaltooligosaccharides (IMOs) have good prebiotic effects, and long IMOs (LIMOs) with a degree of polymerization (DP) of 7 or above show improved effects. However, they are not yet commercially available, and require costly enzymes and processes for production. The N-terminal region of the thermostable Thermoanaerobacter thermocopriae cycloisomaltooligosaccharide glucanotransferase (TtCITase) shows cyclic isomaltooligosaccharide (CI)-producing activity owing to a catalytic domain of glycoside hydrolase (GH) family 66 and carbohydrate-binding module (CBM) 35. In the present study, we elucidated the activity of the C-terminal region of TtCITase (TtCITase-C; Met740-Phe1,559), including a CBM35-like region and the GH family 15 domain. The domain was successfully cloned, expressed, and purified as a single protein with a molecular mass of 115 kDa. TtCITase-C exhibited optimal activity at 40 degrees C and pH 5.5, and retained 100% activity at pH 5.5 after 18-h incubation. TtCITase-C synthesized alpha-1,6 glucosyl products with over seven degrees of polymerization (DP) by an alpha-1,6 glucosyl transfer reaction from maltopentaose, isomaltopentaose, or commercialized maltodextrins as substrates. These results indicate that TtCITase-C could be used for the production of alpha-1,6 glucosyl oligosaccharides with over DP7 (LIMOs) in a more cost-effective manner, without requiring cyclodextran.