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-glucanohydrolase, EC 3.2.1.41) in Bacillus flavocaldarius KP1228 (FERM-P9542) cells growing at 51 to 82 degrees C was expressed in Escherichia coli MV1184. 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 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 (beta/alpha)8-barrel domain, one domain made of the region protruding from the barrel between the third beta-strand and the 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.
Kashiwabara
S
S
Department of Applied Biochemistry, Kyoto Prefectural University, Japan.
Ogawa
S
S
Miyoshi
N
N
Oda
M
M
Suzuki
Y
Y
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D011994:Recombinant Proteins; D004262:DNA Restriction Enzymes; D006026:Glycoside Hydrolases; C100181:pullulanase