title: Site-directed mutagenesis of the conserved threonine, tryptophan, and lysine residues in the starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase.
abstract: The C-terminal domain of Bacillus sp. strain TS-23 alpha-amylase (BLA) has been known to be involved in the raw starch-binding activity of the enzyme. Sequence comparison revealed that Thr-527, Trp-545, Trp-561, Lys-576, and Trp-588 in this domain are highly conserved in the aligned enzymes. To understand structure-function relationships in the starch-binding domain of BLA, site-directed mutagenesis was conducted to replace these residues with leucine or isoleucine. The overexpressed enzymes have been purified by nickel-chelate chromatography, and the molecular mass of the purified proteins was approximately 64.5 kDa. Starch-binding assay showed that the binding activities of the single-mutated enzymes were significantly reduced, while the combinational mutations did not lead to a complete loss of the activity.
authors.affiliation: Department of Food and Nutrition, Hungkuang University, Shalu, Taichung 433, Taiwan.
authors.forename: Huei-Fen
authors.initials: HF
authors.lastname: Lo
authors.forename: Wen-Ying
authors.initials: WY
authors.lastname: Chiang
authors.forename: Meng-Chun
authors.initials: MC
authors.lastname: Chi
authors.forename: Hui-Yu
authors.initials: HY
authors.lastname: Hu
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D013213:Starch / Q000378:metabolism*; D013912:Threonine; D014364:Tryptophan; D000516:alpha-Amylases / Q000737:chemistry*
chemical_list: D013912:Threonine; D014364:Tryptophan; D013213:Starch; D000516:alpha-Amylases; D008239:Lysine