Investigating the structural and functional snapshots of Bacillus licheniformis alpha-amylase through protein engineering strategies.

Alpha-amylases are essential enzymes that cleave α-1,4-glycosidic bonds in starch, generating products such as glucose, maltose, dextrin's, and oligosaccharides, which play a key role in various industries. The structural and functional insights, along with biochemical dynamics, of Bacillus licheniformis α-amylase variants (BLAMWSP and BLAMCD) were investigated through N- and C-terminal truncations along with two previously reported mutations, Thr353Ile and His400Arg. MD simulation results demonstrated the stability of binding and catalytic residues as predicted through molecular docking. Analysis of the secondary structure and temperature ramping through CD spectroscopy revealed that both BLAMWSP and BLAMCD maintained structural stability at 90 °C. The specific activities of BLAMWSP and BLAMCD against wheat starch were determined to be 2343.09 ± 0.20 and 4237.88 ± 0.66 (μmol min/μmole protein), respectively at 90 °C in 100 mM phosphate buffer (pH 6.0). The BLAMCD variant exhibited a two-fold increase in enzymatic activity relative to BLAMWSP due to increased surface accessibility of its substrate-binding and catalytic residues. The enzymes' catalytic efficiencies (k /K) were 51.76 ± 1.76 and 114.10 ± 1.41, highlighting that BLAMCD exhibits significantly higher catalytic efficiency and substrate affinity than BLAMWSP. These modifications make BLAMCD a promising candidate for industrial starch liquefaction and scarification applications.