Journal
PROTEIN AND PEPTIDE LETTERS
Volume 26, Issue 2, Pages 148-157Publisher
BENTHAM SCIENCE PUBL LTD
DOI: 10.2174/0929866526666190116162958
Keywords
alpha-amylase; calcium ions; circular dichroism; differential scanning calorimetry; homology modeling; thermostability
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Funding
- National Natural Science Foundations of China [31560251]
- Natural Science Foundations of Guangxi [2015GXNSFAA139053, 2017GXNSFBA198008]
- Key Project of Guangxi Science & Technology for Research and Development (Guangxi Science and Technology Cooperation Program) [15104001-6]
- basic research program of the Guangxi Academy of Sciences [15YJ22SW02, 2017YJJ23004]
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Background: alpha-Amylases are starch-degrading enzymes and used widely, the study on thermostability of alpha-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 alpha-amylase (AGXA) from thermophilic Anoxybacillus sp.GXS-BL. Methods: alpha-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 (T-opt), half-inactivation temperature (T-50) and thermal inactivation (half-life, t(1/2)) was evaluated. The thermal denaturation of the enzymes was determined by DSC and CD methods. 3D structure of AGXA was homology modeled with a-amylase (5A2A) as the template. Results: With calcium ions, the values of T-opt, T-50, t(1/2), T-m and Delta H in AGXA were significantly higher than those of AGXA without calcium ions, showing calcium ions had stabilizing effects on alpha-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 alpha-helical contents increasing, and the transition from alpha-helices to beta-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.
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