A novel method to produce branched alpha-cyclodextrins: Pullulanase-glucoamylase-mixed method

We have developed a novel method to produce 6-O-alpha-D-glucosyl-alpha-cyclodextrins (G(1)-alpha-CDs) such as G(1)-alpha-CD and 6(1), 6(n)-di-O-alpha-D-glucosyl-alpha-CDs ((G(1))(2)-alpha-CDs). This method is a combined one-step process composed of the condensation of maltose (G(2)) and alpha-CD by reverse-synthesis with pullulanase and hydrolysis of intermediate 6-O-alpha-maltosyl-alpha-CD (G(2)-alpha-CD) by glucoamylase. In a comparison of substrate specificities for three glucoamylases (from Aspergillus niger, Rhizopus niveus, and Endomyces sp.), A. niger glucoamylase had the highest specificity toward G(2)-alpha-CD. The yield of G(1)-alpha-CDs could be increased using this method with A. niger glucoamylase, which suggests that the equilibrium of the condensation reaction with pullulanase slid to G(2)-alpha-CD synthesis as a result of the conversion of G(2)-alpha-CD into G(1)-alpha-CD by glucoamylase. A maximum branched ratio of final products was 69% using this method, whereas that by a conventional method was only 60%. The branched ratio varied under several operational conditions such as total substrate content, initial molar ratio of G(2) to CD (G(2)/CD)i, and enzyme unit ratio of pullulanase to glucoamylase (Up/Ug).