The gene that coded for a cellular pullulanase of type I (α-dextrin 6-glucanohydrolase, EC 3.2.1.41) in Bacillus flavocaldarius KP1228 (FERM-P9542) cells growing at 51 to 82°C was expressed in Escherichia coli MV1184. The enzyme had a half-life of 10 min at 107°C. Purification of the enzyme and its characterization showed that the enzyme was identical with 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 bacterial pullulanases of type I, and of Bacillus stearothermophilus TRS40 neoplullulanase, respectively.
  Sequence analysis showed that B. flavocaldarius pullulanase comprised 3 domains, i.e., one catalytic (β/α)₈-barrel domain, one domain made of the region protruding from the barrel between the third β-strand and the third α-helix, and one β-stranded domain attached to the C-end of the barrel domain, but that the pullulanase lacked the β-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.

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