Deletion analysis of the C-terminal region of the alpha-amylase of Bacillus sp. strain TS-23.
The alpha-amylase from Bacillus sp. strain TS-23 is a secreted starch hydrolase with a domain organization similar to that of other microbial alpha-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 alpha-amylase of Bacillus sp. strain TS-23, four His(6)-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 alpha-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 alpha-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 alpha-amylase is functional and independent from the starch-binding domain.
Lo
Huei-Fen
HF
Department of Food and Nutrition, Hung Kuang Institute of Technology, Shalu, Taichung 433-01, Taiwan.
Lin
Long-Liu
LL
Chiang
Wen-Ying
WY
Chie
Meng-Chun
MC
Hsu
Wen-Hwei
WH
Chang
Chen-Tien
CT
D000327:Adsorption; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013213:Starch / Q000378:metabolism; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D013213:Starch; D000516:alpha-Amylases