Three domains comprised in thermostable molecular weight 54,000 pullulanase of type I from Bacillus flavocaldarius KP1228

The gene that coded for a cellular pullulanase of type I (alpha-dextrin 6-g lucanohydrolase, EC 3.2.1.41) in Bacillus flavocaldarius KP1228 (FERM-P9542 ) cells growing at 51 to 82 degrees C was expressed in Escherichia coli MV1 184. The enzyme had a half-life of 10 min at 107 degrees C. Purification of the enzyme and its characterization showed that the enzyme was identical w ith the native one. Its primary structure of 475 residues with a molecular weight of 53,856 deduced from the gene was 15-21% and 43% identical to the corresponding C-terminal regions in the sequences of 2 plant and 6 bacteria l pullulanases of type I, and of Bacillus stearothermophilus TRS40 neoplull ulanase, respectively. Sequence analysis showed that B. flavocaldarius pullulanase comprised 3 dom ains, i.e., one catalytic (beta/alpha)(g)-barrel domain, one domain made of the region protruding from the barrel between the third beta-strand and th e third alpha-helix, and one beta-stranded domain attached to the C-end of the barrel domain, but that the pullulanase lacked the beta-stranded domain commonly found in addition to the 3 domains in the neopullulanase and all other pullulanases, and attached to the N-end of the barrel domain.