Biochemical characterization and molecular mechanism of acid denaturation of a novel α-amylase from Aspergillus niger

Abstract A novel acid-stable α-amylase from A. niger CBS513.88, AmyE, was purified, characterized, and the molecular mechanism of its acid denaturation was comprehensively analyzed in this study. AmyE exhibited maximal activity at pH 4.2–4.6 and was stable within pH 4.0-5.0. Its optimal temperature was 40 °C and it was stable up to 50 °C. AmyE showed broad substrate specificity, and the main hydrolysis products from soluble starch were glucose, maltose, and maltotriose. Finally, the acid-denaturation mechanism of AmyE was probed using molecular dynamics (MD) simulations. The active site region (ASR) was identified, and was found to include the residues 221–323 which are strongly affected at pH 2.5, especially in the case of Asp313 located in the loop region. The conformational transition of the ASR was accelerated at pH 2.5. The conformational transition of the ASR not only disrupted the distances between the residue Asp313 and the residues Glu246 and Asp222, but also weakened the hydrogen bonds among the residues involved in the ASR, which eventually destroyed the enzyme’s catalytic activity. These MD results explain the molecular mechanism of acid denaturation of AmyE, which will greatly benefit the rational design of more acid-stable α-amylase variants, with potential applications in the baking and starch industries.