The conformational changes in α-amylase induced by different ligands, including metal ions, substrates, and aromatic compounds in liquor production, were systematically studied using spectroscopy. Fluorescence acrylamide quenching analysis showed that the interaction with active metal cations (K+, Na+, and Ca2+) led to higher exposure of the active sites in α-amylase. In contrast, interactions with substrates (soluble starch, amylose, amylopectin, wheat starch, and dextrin) reduced the degree of exposure of active sites, and the conformation of the enzyme became more rigid and compact. Although the interaction with inhibitory metal cations (Mg2+, Zn2+) and aromatic compounds generated in the brewing process (guaiacol, eugenol, thymol, and vanillin) increased the exposure of active site with a relatively low amplitude, it reduced the enzymatic activity. This finding may be due to the overall structure of the enzyme becoming looser. Structural stability showed that the active cations and substrates increased the stability of the secondary structure of the α-amylase backbone, whereas the inhibitory cations and aromatic compounds reduced the stability of the backbone but increased the compact of domain A and B. Enzymatic assays and molecular docking experiments strongly supported these conclusions. The experimental results may provide a valuable reference for controlling related conditions and improving production efficiency.
Keywords: Allosteric regulation; Conformational change; Fluorescence quenching; α-Amylase.
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