Engineered cyclodextrin glucanotransferases from Bacillus sp. G-825-6 produce large-ring cyclodextrins with high specificity

Abstract

Cyclodextrin glucanotransferases (CGTases) synthesize cyclic oligosaccharides (cyclodextrins, CD) from starch. A CGTase from Bacillus sp. G-825-6 was engineered by site-directed mutagenesis at two positions by the construction of the variants Y183W, Y183R, D358R, Y183W/D358R, and Y183R/D358R. Among CD composed of 7–12 glucose units (CD7–CD12), Y183W mainly produced CD8. Y183R had completely lost its ability to synthesize CD7, and CD8 and the larger CD were the only cyclic oligosaccharides produced. D358R also formed mainly CD8–CD12 during a reaction time of 24 hr. The double mutant Y183W/D358R showed combined characteristics of the single mutations with very low CD7 cyclization activity and an increased formation of the larger CD. The results show that CGTases synthesizing mainly CD8–CD12 can be constructed allowing a convenient production of larger CD in significant amounts as host molecules in supramolecular complexing reactions.