Intermolecular transglucosylation of cyclodextrin glycosyltransferase (CGTase) was investigated for its use in oligosaccharide synthesis. From the kinetic parameters of the CGTase-catalyzed transglucosylation reaction, using beta-cyclodextrin (beta-CD) as the glucosyl donor and various saccharides or derivatives as acceptors, the efficient acceptors of the Paenibacillus sp. All enzyme were glucose, sorbose, lactose and cellobiose. Amongst these acceptors, cellobiose showed the highest k(cat)/K-m value. The transglucosylation yields of the reactions for cellobiose, sorbose and glucose acceptors were 78, 57 and 54%, respectively, making cellobiose the most efficient acceptor of the tested saccharides in coupling with beta-CD. The optimal condition for the coupling reaction was determined as: 2%(w/v)beta-CD and 0.5% (w/v)cellobiose, incubated with 64 U/mL of CGTase at 30 degrees C for 2 h. Two main transfer products detected by HPLC, PC1 and PC2, with retention times of 3.81 and 4.42 min, respectively, and a product ratio of 3:1, had a molecular mass of 504 and 666 Da, respectively, as analyzed by mass spectrometry. The structures suggested by NMR were a trisaccharide and a novel tetrasaccharide-containing cellobiose of the structures glc (alpha 1 -> 4) glc (beta 1 -> 4) glc and glc(alpha 1 -> 4) glc (alpha 1 -> 4) glc (beta 1 -> 4) glc, respectively. The products were found to be resistant to hydrolysis by a-amylase. (C) 2010 Elsevier Ltd. All rights reserved.