The action pattern of Bacillus licheniformis thermostable α-amylase (BLA) was analyzed using a series of (14)C-labeled and non-labeled maltooligosaccharides from maltose (G2) to maltododecaose (G12). Maltononaose (G9) was the preferred substrate, and yielded the smallest Km=0.36 mM, the highest kcat=12.86 s(-1), and a kcat/Km value of 35.72 s(-1) mM(-1), producing maltotriose (G3) and maltohexaose (G6) as the major product pair. Maltooctaose (G8) was hydrolyzed into two pairs of products: G3 and maltopentaose (G5), and G2 and G6 with cleavage frequencies of 0.45 and 0.30, respectively. Therefore, we propose a model with nine subsites: six in the terminal non-reducing end-binding site and three at the reducing end-binding site in the binding region of BLA.
