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 amyBL159 from a thermophilic strain Bacillus licheniformis 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 AmyBL159Ec and AmyBL159Bs recombinant α-amylases were purified from Escherichia coli BL21(DE3)pLys and Bacillus subtilis 168 strains, respectively. The AmyBL159Ec enzyme was most active in the range of 75–95 °C, while AmyBL159Bs showed maximum activity at temperatures from 45 to 75 °C. AmyBL159Bs 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 AmyBL159Ec and 142% for AmyBL159Bs). These results highlight the importance of choosing an expression system for modulating the functional characteristics of recombinant α-amylase. The obtained AmyBL159Ec and AmyBL159Bs 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 B. licheniformis identified significant amino acid substitutions at positions 134(133) and 210(209) of the amino acid chain which may contribute to enhanced enzyme thermostability.