Cyclodextrins (CD) are cyclic alpha-1,4-glucans composed of glucose units, and they have multiple applications in food, pharmaceuticals, cosmetics, agriculture, chemicals, etc. CD are usually produced by cyclodextrin glycosyltransferase (CGTase) from starch. In the present study, a simultaneous conversion approach was developed to improve the yield of CD from starch by conjunction use of isoamylase with alpha-CGTase. The isoamylase of Thermobifida fusca was cloned and expressed in Escherichia coli BL21(DE3). The biochemical characterization of the enzyme showed that the optimum temperature and pH of the recombinant enzyme was 50 A degrees C and 5.5, respectively, and it maintained 60 %, 85 % and 78 % relative activity at 30 A degrees C, 40 A degrees C and 60 A degrees C, respectively. When the recombinant isoamylase and alpha-CGTase were used simultaneously to convert potato starch (15 %, w/v) into CD, the optimum conditions were found to be: 10 U of alpha-CGTase and 48 U of isoamylase per gram of substrate, with reaction temperature of 30 A degrees C and pH 5.6. On the optimum condition, the total yield of CD reached 84.6 % (w/w) after 24 h, which was 31.2 % higher than transformation with alpha-CGTase alone. This is the first report of synchronous bioconversion of CD by both alpha-CGTase and isoamylase, and represents the highest efficiency of CD production reported so far.