Structural and functional insights into AmyHa: a haloadapted α-amylase from Haloarcula argentinensis S3 optimized via response surface methodology

The development of robust biocatalysts capable of maintaining structural integrity and catalytic efficiency under high salinity and acidic conditions remains a major challenge in enzyme engineering and industrial starch saccharification. In this study, we combined statistical bioprocess optimization with extensive in silico structural characterization to enhance the production and elucidate the haloadaptive features of an extracellular α-amylase ( AmyHa ) from the extreme haloarchaeon Haloarcula argentinensis strain S3. Media screening using a Plackett–Burman design identified NaCl concentration, soluble starch concentration, and medium pH as the primary factors governing enzyme yield. Subsequent optimization using a Box–Behnken response surface design identified a significant interaction between salinity and pH, indicating that high ionic strength supports enzyme synthesis under acidic conditions. The optimized culture conditions (250 g/L NaCl, 6 g/L soluble starch, and medium adjusted to pH 5.0) yielded a maximum experimental activity of 274.1 ± 1.8 U/mL, representing a 1.84-fold enhancement over the baseline. Primary sequence analysis of the AmyHa protein showed a highly acidic theoretical isoelectric point (pI) of 4.21, attributed to a high proportion of aspartic and glutamic acid residues (18.56%). Topological analyses indicated the absence of canonical N-terminal signal peptides and transmembrane domains, suggesting a potentially non-classical translocation pathway or an alternative initiation sequence. Homology modeling and stereochemical validation confirmed a well-defined tertiary architecture featuring a catalytic core (β/α)8 TIM-barrel domain flanked by domains B and C. Molecular docking simulations indicated strong binding affinity towards branched starch fragments (∆G = -10.0 kcal/mol), mediated by an intricate hydrogen-bonding network surrounding the conserved catalytic triad (Asp197, Glu233, and Asp300). Overall, these findings demonstrate that AmyHa possesses key structural adaptations for extreme environments, offering strong potential for specialized industrial bioprocesses.