Site-directed mutagenesis of the conserved threonine, tryptophan, and lysine residues in the starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase.
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.
Lo
Huei-Fen
HF
Department of Food and Nutrition, Hungkuang University, Shalu, Taichung 433, Taiwan.
Chiang
Wen-Ying
WY
Chi
Meng-Chun
MC
Hu
Hui-Yu
HY
Lin
Long-Liu
LL
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*
D013912:Threonine; D014364:Tryptophan; D013213:Starch; D000516:alpha-Amylases; D008239:Lysine