Di-O-alpha-maltosyl-beta-cyclodextrin ((G2)(2)-beta-CD) was synthesized from 6-O-alpha-maltosyl-beta-cyclodextrin (G2-beta-CD) via a transglycosylation reaction catalyzed by TreX, a debranching enzyme from Sulfolobus solfataricus P2. TreX showed no activity toward glucosyl-beta-CD, but a transfer product (1) was detected when the enzyme was incubated with maltosyl-beta-CD, indicating specificity for a branched glucosyl chain bigger than DP2. Analysis of the structure of the transfer product (1) using MALDI-TOF/MS and isoamylase or glucoamylase treatment revealed it to be dimaltosyl-beta-CD, suggesting that TreX transferred the maltosyl residue of a G2-beta-CD to another molecule of G2-beta-CD by forming an alpha-1,6-glucosidic linkage. When [(14)C]-maltose and maltosyl-beta-CD were reacted with the enzyme, the radiogram showed no labeled dimaltosyl-beta-CD; no condensation product between the two substrates was detected, indicating that the synthesis of dimaltosyl-beta-CD occurred exclusively via transglycosylation of an alpha-1,6-glucosidic linkage. Based on the HPLC elution profile, the transfer product (1) was identified to be isomers of 6(1),6(3)- and 6(1),6(4)-dimaltosyl-beta-CD. Inhibition studies with beta-CD on the transglycosylation activity revealed that beta-CD was a mixed-type inhibitor, with a K(i) value of 55.6 micromol/mL. Thus, dimaltosyl-beta-CD can be more efficiently synthesized by a transglycosylation reaction with TreX in the absence of beta-CD. Our findings suggest that the high yield of (G2)(2)-beta-CD from G2-beta-CD was based on both the transglycosylation action mode and elimination of the inhibitory effect of beta-CD.