alpha-Glucan phosphorylase (GP, EC 2.4.1.1) catalyzes the reversible phosphorolysis of glucan and is considered to play a central role. in the mobilization of carbohydrate reserves. The structural gene for GP has already been cloned and sequenced from Bacillus stearothermophilus TRBE14 [Takata et al., J. Bacteriol., 179, 4689-4698, 1997; DDBJ/EMBL/GenBank accession No. D87026]. This enzyme was expressed in Escherichia coil and purified to homogeneity, and its enzymatic properties were analyzed. At pH 7, GP was stable up to 40 degrees C, and the optimum temperature for activity was 50 degrees C. The enzyme was stable in the pH range 6.5 to 11, and the optimum pH was 7. A test of substrate preference demonstrated that starch and glycogen were more reactive substrates than linear maltodextrin. The smallest acceptor for a synthetic reaction was maltotetraose (G4), and G4 was left as a final product after the phosphorolysis of a linear saccharide. ADP-glucose and UDP-glucose strongly inhibited this enzyme activity. Although the substrate specificity and type of inhibitors are similar to those of its counterpart from E. coli, B. stearothermophilus GP had about a 100-fold higher affinity, and much higher specific activity for glycogen than the E. coli enzyme. These results suggest that the rates of glycogen degradation in the two species may be different from each other.