Comparison of starch hydrolysis activity and thermal stability of two Bacillus licheniformis alpha-amylases and insights into engineering alpha-amylase variants active under acidic conditions

Bacillus licheniformis alpha-amylase (BLA) is widely used in various procedures of starch degradation in the food industry, and a BLA species with improved activity at higher temperature and under acidic conditions is desirable. Two BLA species, designated as PA and MA, have been isolated from the wild-type B. licheniformis strain and a mutant strain, respectively. In this study, their starch-hydrolysis activity and thermal stability were examined. MA showed higher activity than PA, especially at acidic pH (pH 5.0-5.5), and even after 1 h of treatment at 90 degrees C. MA was active in the range of pH 4.0-8.0, which is much wider than that (pH 4.5-7.5) of PA. It was shown that the proton dissociation constants on the acidic and alkaline sides (pKa1 and pKa2) were shifted to more acidic and basic values, respectively, by the mutation of PA to MA. The activation energy and thermodynamic parameters for their thermal inactivation indicate that MA is more thermally stable and catalytically active than PA, suggesting that MA could be useful for glucose-production process coupled with reactions catalyzed by beta-amylase.