CLUSTERED PROLINE RESIDUES AROUND THE ACTIVE-SITE CLEFT IN THERMOSTABLE OLIGO-1,6-GLUCOSIDASE OF BACILLUS-FLAVOCALDARIUS KP1228

The gene that coded for a cellular oligo-1,6-glucosidase (dextrin 6-al pha-D-glucanohydrolase, EC 3.2.1.10) in Bacillus flavocaldarius KP1228 (FERM-P9542) cells growing at 51-82 degrees C was expressed in Escher ichia coli JM109. The enzyme had a half-life of 10 min at 89.2 degrees C. Purification of the enzyme and its characterization showed that th e enzyme was identical with the native one. Its primary structure of 5 29 residues with a molecular weight of 61,469 deduced from the gene wa s 40-42 % identical to the sequences of less thermostable oligo-1,6-gl ucosidases from Bacillus cereus ATCC 7064, Bacillus coagulans ATCC 705 0, and Bacillus thermoglucosidasius KP1006. Sequence analysis showed t hat the B. flavocaldarius enzyme shared 14 proline residues at the sam e positions as in the three other enzymes, and that the B. flavocaldar ius enzyme had 22 of 33 additional proline residues (cf, 1/5, 5/10, an d 9/18 in the respective counterparts) in three long polypeptides cons tituting the active-site cleft, which connected the third, fourth, and eighth beta-strands to the corresponding third, fourth, and eighth al pha-helices in the (beta/alpha)(8)-barrel.