The thermoalkaliphilic anaerobic bacterium Anaerobranca gottschalkii produces an extracellular CGTase when grown on starch at 55 degrees C and pH 9.0. The gene encoding this CGTase was cloned and successfully expressed in Escherichia coli. It encodes a protein consisting of 721 amino acids with a signal sequence of 34 amino acids. On SDS-polyacrylamide gels, the purified CGTase from A. gottschalkii displayed the expected molecular mass of 78 kDa. The recombinant enzyme was purified with a yield of 13.5% and displayed a specific activity of 210 units/mg. This CGTase, which represents the first report of a CGTase from an anaerobic thermoalkaliphile, was active at a broad range of temperature and pH, namely 55-70 degrees C and pH 5-10. It completely converted amylose, amylopectin and native starch to cyclodextrins, preferentially alpha-cyclodextrin. With a longer incubation period, the alpha-cyclodextrin to beta-cyclodextrin ratio declined. Variations in substrate type and concentration influenced the product pattern. Increasing the substrate concentration (0.5-20.0%) and glucans containing branching points (alpha-1,6 glycosidic linkages) shifted the product pattern to: beta-cyclodextin > alpha-cyclodextrin > gamma-cyclodextrin. In addition to these cyclodextrins, larger cyclodextrins (>8 glucose units) were formed in the initial reaction period. The CGTase was stabilised against thermal inactivation by calcium ions and high substrate concentrations; and 5 mM of CaCl(2) shifted the apparent melting point of the enzyme from 60 degrees C to 69 degrees C.