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.