RESUMO
BaqA is a raw starch degrading α-amylase produced by the marine bacterium Bacillus aquimaris MKSC 6.2, associated with soft corals. This α-amylase belongs to a new subfamily Glycoside Hydrolases (GH) 13_45 which has several unique characteristics, namely, a pair of tryptophan residues Trp201 and Trp202, a distinct LPDIx signature in the Conserved Sequence Region-V (CSR-V), and an elongated C-terminus containing five aromatic residues. The research aims to investigate physicochemical, kinetics, and biochemical properties of BaqA. In this study, the full-length enzyme (BaqA) and a truncated form of BaqA (designated as BaqAΔC), lacking the C-terminal 34 amino acids were constructed and expressed in Escherichia coli ArcticExpress (DE3). BaqA formed inclusion bodies, while BaqAΔC was produced as a soluble protein. Purified and refolded BaqA exhibited a catalytic efficiency (k cat/K m) of 53.1 ± 6.3 mL mg-1 s-1 at 40 °C and pH 7.5, whereas the purified BaqAΔC displayed k cat/K m of 11.4 ± 1.3 mL mg-1 s-1 under the optimum condition of 50 °C and pH 6.5. Moreover, BaqAΔC showed a slight reduction in the binding affinity towards sago granules. Interestingly, BaqAΔC displayed robust stability and halotolerant properties compared to BaqA. BaqAΔC maintained 50 % amylolytic activity for up to 6 h, whereas BaqA lost over 50 % of its activity within 90 min. Furthermore, BaqAΔC showed a remarkable increase in amylolytic activity upon the addition of NaCl, with an optimum concentration of 0.5 M. Even at a high salt concentration (1.5 M NaCl), BaqAΔC retained over 50 % of its residual activity. Taken together, its solubility, amylolytic activity, stability, ability to degrade raw starch, and moderate halotolerance make BaqAΔC a promising candidate for various starch processing industries.
RESUMO
The α-amylase BmaN1 from Bacillus megaterium 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 Escherichia coli 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 B. megaterium 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.