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.
Keywords: Bacillus; cyclodextrin glucanotransferase; cyclodextrins; protein engineering; side-directed mutagenesis.
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