Bacillus amyloliquefaciens alpha-amylase (BAA) is an industrially important extra cellular enzyme with a number of applications. The acid-induced unfolding of BAA was studied by far- and near-UV circular dichroism (CD), intrinsic fluorescent emission spectroscopy, 1-anilino naphthalene-8-sulfonate (ANS) binding, light scattering and fluorescence quenching by acrylamide. At pH 3.0, BAA acquires partially folded state, which shows characteristics of molten globules, i.e., reduction of defined tertiary structure and almost no change in the secondary structure. ANS binding and light scattering experiments show that at acidic pH, BAA unfolds in such a way that its hydrophobic surface is exposed to a greater extent compared to the native form. In addition, acrylamide quenching of the intrinsic tryptophan residues in the protein molecules indicate that at pH 3.0, the protein is in a partially unfolded conformation with more tryptophan residues exposed to the solvent as compared to the native conformation in the neutral pH. Calcium ion is required for the refolding of the molten globule state to the native form. Enzymatic activity is recovered in the presence of Ca2+ but no reactivation of the enzyme can be observed in the presence of Mg2+, Mn2+ and EDTA. Based on several lines of evidence, it is proposed that such acid-denatured state may be the competent state involved in the secretion process of alpha-amylase. (C) 2004 Elsevier Inc. All rights reserved.