title: Biochemical characterization of two truncated forms of amylopullulanase from Thermoanaerobacterium saccharolyticum NTOU1 to identify its enzymatically active region.
abstract: The enzymatically active region of amylopullulanase from Thermoanaerobacterium saccharolyticum NTOU1 (TsaNTOU1Apu) was identified by truncation mutagenesis. Two truncated TsaNTOU1Apu enzymes, TsaNTOU1ApuM957 and TsaNTOU1ApuK885, were selected and characterized. Both TsaNTOU1ApuM957 and TsaNTOU1ApuK885 showed similar specific activities toward various substrates. The overall catalytic efficiency (k (cat)/apparent K (m)) for the soluble starch or pullulan substrate, however, was 20-25% lower in TsaNTOU1ApuK885 than in TsaNTOU1ApuM957. Both truncated enzymes exhibited similar thermostability and substrate-binding ability against the raw starch. The fluorescence and circular dichroism spectrometry studies indicated that TsaNTOU1ApuK885 retained an active folding conformation similar to that of TsaNTOU1ApuM957. These results indicate that a large part of the TsaNTOU1Apu, such as the C-terminal carbohydrate-binding module family 20, the second fibronectin type III, and a portion of the first FnIII motifs, could be removed without causing a serious aberrant structural change or a dramatic decrease in hydrolysis of soluble starch and pullulan.
authors.affiliation: Department of Life Science, National Taiwan Ocean University, Keelung, Taiwan. fpl5505@mail.ntou.edu.tw
authors.forename: Fu-Pang
authors.initials: FP
authors.lastname: Lin
authors.forename: Hsiu-Yen
authors.initials: HY
authors.lastname: Ma
authors.forename: Hui-Ju
authors.initials: HJ
authors.lastname: Lin
authors.forename: Shiu-Mei
authors.initials: SM
authors.lastname: Liu
authors.forename: Wen-Shyong
authors.initials: WS
authors.lastname: Tzou
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005936:Glucans / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006868:Hydrolysis; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D010957:Plasmids; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013213:Starch / Q000378:metabolism; D013379:Substrate Specificity; D046950:Thermoanaerobacterium / Q000201:enzymology* / Q000235:genetics; D014169:Transformation, Bacterial
chemical_list: D001426:Bacterial Proteins; D005936:Glucans; D007527:Isoenzymes; D011994:Recombinant Proteins; C009109:pullulan; D013213:Starch; D006026:Glycoside Hydrolases; C082507:amylopullulanase