C-TERMINAL TRUNCATIONS OF A THERMOSTABLE BACILLUS-STEAROTHERMOPHILUS ALPHA-AMYLASE

A series of truncated proteins from a thermostable Bacillus stearother mophilus alpha-amylase was prepared to study the importance of the ext ension 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 speci fic activity of the enzyme. Only the two longest mutant proteins were active: the specific activity of the 498 residue variant was 97% and p rotein 483 was 36% that of the parental enzyme. The K-m values of star ch hydrolysis changed from 1.09 for wild type enzyme to 0.35 and 0.21 for mutants 498 and 483, respectively, indicating altered substrate bi nding. The mutant enzymes had almost identical pH and temperature opti ma with the wild type amylase, but enhanced thermal stability and alte red end product profile, The consequences of the truncation to the str ucture and function of the enzymes were explored with molecular modeli ng, The liquefying amylases seem to require similar to 480 residues to be active, whereas the C-terminal end of B.stearothermophilus amylase is required for increased activity.