Screening, Gene Cloning, and Characterizations of an Acid-Stable alpha-Amylase

Based on its alpha-amylase activity at pH 5.0 and optimal pH of the crude enzyme, a strain (named B-5) with acid alpha-amylase production was screened. The B-5 strain was identified as Bacillus amyloliquefaciens through morphological, physiological, and biochemical characteristics analysis, as well as 16S rDNA phylogenetic analysis. Its alpha-amylase gene of GenBank Accession No. GU318401 was cloned and expressed in Escherichia coli. The purified recombinant alpha-amylase AMY-Ba showed the optimal pH of 5.0, and was stable at a pH range of 4.0-6.0. When hydrolyzing soluble starch, amylose, and amylopectin, AMY-Ba released glucose and maltose as major end products. The vamylase AMY-Ba in this work was different from the well-investigated J01542-type alpha-amylase which also came from B. amyloliquefaciens. AMY-Ba exhibited notable adsorption and hydrolysis ability towards various raw starches. Structure analysis of AMY-Ba suggested the presence of a new starch-binding domain at its C-terminal region.