Molecular cloning and modelling of a thermostable α-amylase from a thermophilic Geobacillus sp. DS3.

Thermostable α-amylase from Geobacillus sp. DS3, isolated from the Sikidang Crater, Dieng Plateau, Indonesia, was previously purified and characterized. However, production from thermophilic bacteria requires high-temperature cultivation. This study aimed to clone and express the amy gene encoding α-amylase in Escherichia coli BL21(DE3) for easier enzyme production. The amy gene (1638 bp) was amplified via PCR, TA-cloned, and inserted into the pET-SUMO expression vector, which includes an N-terminal His-tag and SUMO-tag to enhance expression and solubility. The recombinant plasmid (pET-SUMO-amy) was transformed into E. coli BL21(DE3) for protein expression. Homology modelling using MOE software and template PDB ID 1HVX (91.5% identity) revealed a reliable 3D structure. Structural analysis showed altered calcium and sodium ion binding compared to the template, with calcium ions interacting with more residues. Docking studies revealed that maltotetraose binding is stabilized by five key residues: Asp268, His272, Trp300, Asn363, and Asp365. The enzyme displayed optimal activity at 70 °C and retained 60% activity at 90 °C. Kinetic parameters showed a low K_m (6.77 mM) and V_max (0.20 U/mL), indicating high substrate affinity. In conclusion, the recombinant α-amylase exhibited thermostability and substrate affinity suitable for industrial applications such as starch liquefaction and porous starch production at elevated temperatures.