Recombinant Forms of α-Amylase AmyBL159 from a Thermophilic Bacterium Bacillus licheniformis MGMM159: The Effect of the Expression System on the Enzyme Properties

We present the cloning and heterologous expression of the α-amylase gene <i>amy</i>BL159 from a thermophilic strain <i>Bacillus licheniformis</i> MGMM159, which was isolated from wastewater sediments self-heated to 70 °C. The gene was successfully cloned into the pET22b and pHT01 vectors, expressed and AmyBL159_Ec and AmyBL159_Bs recombinant α-amylases were purified from <i>Escherichia coli</i> BL21(DE3)pLys and <i>Bacillus subtilis</i> 168 strains, respectively. The AmyBL159_Ec enzyme was most active in the range of 75-95 °C, while AmyBL159_Bs showed maximum activity at temperatures from 45 to 75 °C. AmyBL159_Bs was shown to be more thermostable. Both enzymes were active over a broad pH range of 4.0-12.0. It was shown that Mn²⁺ ions enhanced the activity of both enzymes (up to 163% for AmyBL159_Ec and 142% for AmyBL159_Bs). These results highlight the importance of choosing an expression system for modulating the functional characteristics of recombinant α-amylase. The obtained AmyBL159_Ec and AmyBL159_Bs enzymes are promising for biotechnological applications under extreme conditions. The structure of the α-amylase was generated using the AlphaFold 3 web service. A structure-function analysis of this enzyme and previously studied α-amylases from <i>B. licheniformis</i> identified significant amino acid substitutions at positions 134(133) and 210(209) of the amino acid chain which may contribute to enhanced enzyme thermostability.