title: Novel characteristics of a carbohydrate-binding module 20 from hyperthermophilic bacterium.
abstract: In this study, a gene fragment coding carbohydrate-binding module 20 (CBM20) in the amylopullulanase (APU) gene was cloned from the hyperthermophilic bacteria Thermoanaerobacter pseudoethanolicus 39E and expressed in Escherichia coli. The protein, hereafter Tp39E, possesses very low sequence similarity with the CBM20s previously reported and has no starch binding site 2. Tp39E did not demonstrate thermal denaturation at 50 °C; however, thermal unfolding of the protein was observed at 59.5 °C. A binding assay with Tp39E was conducted using various soluble and insoluble substrates, and starch was the best binding polysaccharide. Intriguingly, Tp39E bound, to a lesser extent, to soluble and insoluble xylan as well. The dissociation constant (K d) and the maximum specific binding (B max) of Tp39E to corn starch granules were 0.537 μM and 5.79 μM/g, respectively, at pH 5.5 and 20 °C. 99APU1357 with a Tp39E domain exhibited 2.2-fold greater activity than a CBM20-truncation mutant when starch granules were the substrate. Tp39E was an independently thermostable CBM and had a considerable effect on APU activity in the hydrolysis of insoluble substrates.
authors.affiliation: Department of Food Science and Technology, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon, 305-764, Korea.
authors.forename: Il-Nam
authors.initials: IN
authors.lastname: Oh
authors.forename: Jay-Lin
authors.initials: JL
authors.lastname: Jane
authors.forename: Kan
authors.initials: K
authors.lastname: Wang
authors.forename: Jong-Tae
authors.initials: JT
authors.lastname: Park
authors.forename: Kwan-Hwa
authors.initials: KH
authors.lastname: Park
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D013379:Substrate Specificity; D046968:Thermoanaerobacter / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D006026:Glycoside Hydrolases; C082507:amylopullulanase