Probing the function of C-terminal region of recombinant α-amylase BmaN1 from <i>Bacillus megaterium</i> NL3

The α-amylase BmaN1 from <i>Bacillus megaterium</i> NL3 is a member of GH13_45 subfamily that has a conserved C-terminal region of approximately 30 residues. This region features a motif of five aromatic amino acids predicted to play a role in starch binding. This study aimed to unravel the role of the C-terminal region in starch hydrolysis. The full-length and C-terminally truncated forms of BmaN1 (BmaN1∆C) were expressed in <i>Escherichia coli</i> ArcticExpress (DE3), resulting in proteins with molecular weights of 56 kDa and 49 kDa, respectively. They exhibited comparable enzymatic activity in the hydrolysis of soluble starch, displaying versatility across a wide range of pH values, temperatures, and NaCl concentrations. BmaN1 and BmaN1∆C activities were inhibited by acarbose and were reduced by SDS and EDTA. In terms of binding and degrading the starch granules, BmaN1∆C showed lower affinity and activity in comparison to BmaN1. Our study indicates that the C-terminal region of BmaN1 significantly enhances its binding affinity and degrading the raw starches.IMPORTANCEα-Amylase (EC 3.2.1.1) stands as an endo-acting enzyme, essential for catalyzing the hydrolysis of α-1,4 glycosidic bonds within starch molecules. The relevance of α-amylases in biotechnological applications is substantial, constituting approximately 30% of the global enzyme market. Among these enzymes, BmaN1 was the first α-amylase identified to possess distinct catalytic residues within the GH13 family. BmaN1 from <i>B. megaterium</i> NL3 belongs to the GH13_45 subfamily. This subfamily is characterized by a conserved C-terminal region consisting of approximately 30 residues that contains a motif of five aromatic residues predicted to be involved in starch binding. Our study shows that the C-terminal effectively contributes to binding and degrading the raw starch granules. This pioneering research on BmaN1 expands our understanding of α-amylases and holds promise for innovative biotechnological advancements.