Coupling Reaction of Bacillus macerans Cyclodextrin Glucanotransferase on Glucosyl-α-cyclodextrin and Glucose

Treatment of glucosyl-α-cyclodextrin with radioactive glucose in the presence of Bacillus macerans cyclodextrin glucanotransferase, yielded radioactive maltose, maltotriose, and four kinds of glucosyl branched oligosaccharides. The branched oligosaccharides formed had identical Rf values as those of branched oligosaccharides of 5-8 glucose unit on paper chromatography using the solvent system of 1-butanol: pyridine: water (6/4/4), (Fractions B₅-B₈). Individual oligosaccharides were isolated and their structures determined using the action of porcine pancreatic α-amylase, β-amylase, pullulanase, isopullulanase and glucoamylase. The structure of the main component of the fraction B₅ was 6⁴-O-α-glucosylmaltotetraose, B₆ was 6⁴-O-α-glucosylmatopentaose, B₇ was 6⁵-O-α-glucosylmaltohexaose, and B₈ was 6⁶-O-α-glucosylmaltoheptaose. Fraction B₈, which formed at the initial stage of the reaction, contained 6⁴, 6⁵ and 6⁶-O-α-glucosylmaltoheptaose. Reducing end ¹⁴C-labelled 6⁴-O-α-glucosylmaltotetraose was degraded to 6³-O-α-glucosylmaltotriose and radioactive glucose by the exhaustive action of Bacillus macerans cycloamylose glucanotransferase, and 6³-O-α-glucosylmaltotriose was completely non-reactive to the enzyme. From these results, we proposed a model of the active site of the enzyme.