PURIFICATION AND CHARACTERIZATION OF ALPHA-GLUCAN PHOSPHORYLASE FROM BACILLUS-STEAROTHERMOPHILUS

alpha-Glucan phosphorylase (GP, EC 2.4.1.1) catalyzes the reversible p hosphorolysis of glucan and is considered to play a central role. in t he mobilization of carbohydrate reserves. The structural gene for GP h as 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 Escherichi a 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 t emperature 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 pre ference demonstrated that starch and glycogen were more reactive subst rates than linear maltodextrin. The smallest acceptor for a synthetic reaction was maltotetraose (G4), and G4 was left as a final product af ter the phosphorolysis of a linear saccharide. ADP-glucose and UDP-glu cose strongly inhibited this enzyme activity. Although the substrate s pecificity and type of inhibitors are similar to those of its counterp art 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 degradat ion in the two species may be different from each other.