Molecular Cloning and Biochemical Characterization of a novel thermostable α-amylase of Bacillus subtilis MDC 3500 isolated from acidic soils in Armenia

Amylases are one of the most important industrial enzymes, accounting for 30% of the world's production of enzymes. The quest for novel recombinant α-amylases with enhanced traits remains a pressing challenge, presenting continual relevance in biotechnological sectors. Bacillus subtilis strain MDC 3500 was isolated in acidic soils (pH 3.5-4.0) of Armenia. In this study, the α-amylase gene of Bacillus subtilis MDC 3500 (AmyBS) was cloned by the golden-gate cloning technique followed by intracellular expression in Escherichia coli cells. Phylogenetic analysis revealed a close relationship between AmyBS and α-amylases of Bacillus subtilis A28, exhibiting 97.7% homology. AmyBS was expressed and purified to homogeneity using a two-step purification process involving immobilized metal affinity chromatography and size exclusion chromatography. The temperature and pH optimum, thermal stability, and several other catalytic characteristics of AmyBS were studied. The enzyme exhibits the following order of starch substrate preference: potato > wheat > corn > rice. AmyBS also exhibits specificity for amylose, amylopectin, γ-cyclodextrin, and β-cyclodextrin in decreasing order. The hydrolytic products of potato, corn, or rice starches mainly lead to the accumulation of glucose, maltose, and, to a lesser extent, maltotriose in the reaction medium.