A series of truncated proteins from a thermostable Bacillus stearothermophilus alpha-amylase was prepared to study the importance of the extension in the C-terminus compared with other liquefying Bacillus alpha-amylases. The mutations introducing new translation termination sites shortened the 515 amino acid residue-long wild type enzyme by 17, 32, 47, 73 or 93 residues. The longer the truncation, the lower the specific activity of the enzyme. Only the two longest mutant proteins were active: the specific activity of the 498 residue variant was 97% and protein 483 was 36% that of the parental enzyme. The Km values of starch hydrolysis changed from 1.09 for wild type enzyme to 0.35 and 0.21 for mutants 498 and 483, respectively, indicating altered substrate binding. The mutant enzymes had almost identical pH and temperature optima with the wild type amylase, but enhanced thermal stability and altered end product profile. The consequences of the truncation to the structure and function of the enzymes were explored with molecular modeling. The liquefying amylases seem to require approximately 480 residues to be active, whereas the C-terminal end of B.stearothermophilus amylase is required for increased activity.
Amino Acid Sequence Bacillus