Deletion analysis of the C-terminal region of the α-amylase of Bacillus sp. strain TS-23

Abstract.

The α-amylase from Bacillus sp. strain TS-23 is a secreted starch hydrolase with a domain organization similar to that of other microbial α-amylases and an additional functionally unknown domain (amino acids 517–613) in the C-terminal region. By sequence comparison, we found that this latter domain contained a sequence motif typical for raw-starch binding. To investigate the functional role of the C-terminal region of the α-amylase of Bacillus sp. strain TS-23, four His₆-tagged mutants with extensive deletions in this region were constructed and expressed in Escherichia coli. SDS-PAGE and activity staining analyses showed that the N- and C-terminally truncated α-amylases had molecular masses of approximately 65, 58, 54, and 49 kDa. Progressive loss of raw-starch-binding activity occurred upon removal of C-terminal amino acid residues, indicating the requirement for the entire region in formation of a functional starch-binding domain. Up to 98 amino acids from the C-terminal end of the α-amylase could be deleted without significant effect on the raw-starch hydrolytic activity or thermal stability. Furthermore, the active mutants hydrolyzed raw corn starch to produce maltopentaose as the main product, suggesting that the raw-starch hydrolytic activity of the Bacillus sp. strain TS-23 α-amylase is functional and independent from the starch-binding domain.