Clustered Proline Residues around the Active-Site Cleft in Thermostable Oligo-1,6-glucosidase of Bacillus flavocaldarius KP1228

Abstract

The gene that coded for a cellular oligo-1,6-glucosidase (dextrin 6-α-D-glucanohydrolase, EC 3.2.1.10) in Bacillus flavocaldarius KP1228 (FERM-P9542) cells growing at 51-82°C was expressed in Escherichia coli JM109. The enzyme had a half-life of 10 min at 89.2°C. Purification of the enzyme and its characterization showed that the enzyme was identical with the native one. Its primary structure of 529 residues with a molecular weight of 61,469 deduced from the gene was 40-42% identical to the sequences of less thermostable oligo-1,6-glucosidases from Bacillus cereus ATCC 7064, Bacillus coagulans ATCC 7050, and Bacillus thermoglucosidasius KP1006.

Sequence analysis showed that the B. flavocaldarius enzyme shared 14 proline residues at the same positions as in the three other enzymes, and that the B. flavocaldarius enzyme had 22 of 33 additional proline residues (cf. 1/5, 5/10, and 9/18 in the respective counterparts) in three long polypeptides constituting the active-site cleft, which connected the third, fourth, and eighth β-strands to the corresponding third, fourth, and eighth α-helices in the (β/α)₈-barrel.

• thermostable oligo-1,6-glucosidase
• gene cloning
• Bacillus flavocaldarius
• proline sites
• thermostability