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.

Large‐Ring Cyclodextrins as Chiral Selectors for Enantiomeric Pharmaceuticals

“…At initial reaction conditions, CGTases produce CD of a wide size spectrum . Larger CD are rapidly degraded by the enzyme in a reverse reaction to linear glucans which are subsequently converted to smaller CD . Mixtures of mostly CD6, CD7, and CD8 are therefore the final products of the CGTase reaction with starch while larger CD with a DP of 9 and above can only be obtained in very small quantities .…”

“…The use of the CGTase G825 variant Y183R engineered to specifically produce larger CD allowed us to prepare large‐ring CD in sufficient amounts for further analysis . Since the CGTase synthesized CD8 as the smallest cyclic product, a high proportion of larger CD in the reaction mixtures could be obtained facilitating their subsequent purification . The very small amounts of large‐ring CD produced by commercially available CGTases has been a major bottleneck in the study of these novel supramolecular host compounds.…”

Large‐Ring Cyclodextrins as Chiral Selectors for Enantiomeric Pharmaceuticals

“…[11] Larger CD are rapidly degraded by the enzyme in ar everse reaction to linear glucans which are subsequently converted to smaller CD. [11][12][13] Mixtures of mostly CD6, CD7, and CD8 are therefore the final products of the CGTase reaction with starch while larger CD with aDP of 9a nd above can only be obtained in very small quantities. [4,13] Owing to the lack of efficient synthesis and separation techniques,o nly limited information has been available on the properties of the larger CD as supramolecular host compounds.…”

“…Theuse of the CGTase G825 variant Y183R engineered to specifically produce larger CD allowed us to prepare largering CD in sufficient amounts for further analysis. [12] Since the CGTase synthesized CD8 as the smallest cyclic product, ahigh proportion of larger CD in the reaction mixtures could be obtained facilitating their subsequent purification. [12] The very small amounts of large-ring CD produced by commercially available CGTases has been am ajor bottleneck in the study of these novel supramolecular host compounds.U eda and co-workers isolated CD9 and larger CD from acommercial CD mixture which involved the degradation of the noncyclic glucans by yeast and the addition of complexing agents to remove CD6-CD8, resulting in ayield of only 0.03 %CD9 and 0.01 %C D12.…”

“…[12] Since the CGTase synthesized CD8 as the smallest cyclic product, ahigh proportion of larger CD in the reaction mixtures could be obtained facilitating their subsequent purification. [12] The very small amounts of large-ring CD produced by commercially available CGTases has been am ajor bottleneck in the study of these novel supramolecular host compounds.U eda and co-workers isolated CD9 and larger CD from acommercial CD mixture which involved the degradation of the noncyclic glucans by yeast and the addition of complexing agents to remove CD6-CD8, resulting in ayield of only 0.03 %CD9 and 0.01 %C D12. [7,14] By at reatment with glucoamylase,w e could efficiently degrade residual linear glucans and remove most of the glucose in the reaction mixture by as imple precipitation step,avoiding the use of complexing agents.…”