Background: α-Amylases are starch-degrading enzymes and used widely, the study on
thermostability of α-amylase is a central requirement for its application in life science and biotechnology.
Objective: In this article, our motivation is to study how the effect of Ca2+ ions on the structure and
thermal characterization of α-amylase (AGXA) from thermophilic Anoxybacillus sp.GXS-BL.
Methods: α-Amylase activity was assayed with soluble starch as the substrate, and the amount of
sugar released was determined by DNS method. For AGXA with calcium ions and without calcium
ions, optimum temperature (Topt), half-inactivation temperature (T50) and thermal inactivation (halflife,
t1/2) was evaluated. The thermal denaturation of the enzymes was determined by DSC and CD
methods. 3D structure of AGXA was homology modeled with α-amylase (5A2A) as the template.
Results: With calcium ions, the values of Topt, T50, t1/2, Tm and ΔH in AGXA were significantly
higher than those of AGXA without calcium ions, showing calcium ions had stabilizing effects on
α-amylase structure with the increased temperature. Based on DSC measurements AGXA underwent
thermal denaturation by adopting two-state irreversible unfolding processes. Based on the CD
spectra, AGXA without calcium ions exhibited two transition states upon unfolding, including α-
helical contents increasing, and the transition from α-helices to β-sheet structures, which was obviously
different in AGXA with Ca2+ ions, and up to 4 Ca2+ ions were located on the inter-domain or
intra-domain regions according to the modeling structure.
Conclusion: These results reveal that Ca2+ ions have pronounced influences on the thermostability
of AGXA structure.