title: Fusion of Bacillus stearothermophilus leucine aminopeptidase II with the raw-starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase generates a chimeric enzyme with enhanced thermostability and catalytic activity.
abstract: Bacillus stearothermophilus leucine aminopeptidase II (LAPII) was fused at its C-terminal end with the raw-starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase. The chimeric enzyme (LAPsbd), with an apparent molecular mass of approximately 61 kDa, was overexpressed in IPTG-induced Escherichia coli cells and purified to homogeneity by nickel-chelate chromatography. The purified enzyme retained LAP activity and adsorbed raw starch. LAPsbd was stable at 70 degrees C for 10 min, while the activity of wild-type enzyme was completely abolished under the same environmental condition. Compared with the wild-type enzyme, the twofold increase in the catalytic efficiency for LAPsbd was due to a 218% increase in the k(cat) value.
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 University Road, 60083, Chiayi, Taiwan.
authors.forename: Yu-Wen
authors.initials: YW
authors.lastname: Hua
authors.forename: Meng-Chun
authors.initials: MC
authors.lastname: Chi
authors.forename: Huei-Fen
authors.initials: HF
authors.lastname: Lo
authors.forename: Wen-Hwei
authors.initials: WH
authors.lastname: Hsu
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
mesh_terms: D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D007931:Leucyl Aminopeptidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013213:Starch / Q000378:metabolism; D013696:Temperature; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D013213:Starch; D000516:alpha-Amylases; D007931:Leucyl Aminopeptidase