Liao Si-Ming, Liang Ge, Zhu Jing, Lu Bo, Peng Li-Xin, Wang Qing-Yan, Wei Yu-Tuo, Zhou Guo-Ping, Huang Ri-Bo
National Engineering Research Center for Non-food Biorefinery, State Key Laboratory of Non-food Biomass and Enzyme Technology, Guangxi Key Laboratory of Bio-refinery, Guangxi Academy of Sciences, 98 Daling Road, Nanning, 530007, China.
College of Life Science and Technology, Guangxi University, Nanning, Guangxi, 530004, China.
Protein Pept Lett. 2019;26(2):148-157. doi: 10.2174/0929866526666190116162958.
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.
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.
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.
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.
These results reveal that Ca2+ ions have pronounced influences on the thermostability of AGXA structure.
α-淀粉酶是淀粉降解酶，应用广泛，对其热稳定性的研究是其在生命科学和生物技术中应用的核心要求。
本文旨在研究钙离子对嗜热栖热放线菌GXS-BL来源的α-淀粉酶（AGXA）结构和热特性的影响。
以可溶性淀粉为底物测定α-淀粉酶活性，采用DNS法测定释放的糖量。评估有钙离子和无钙离子的AGXA的最适温度（Topt）、半失活温度（T50）和热失活（半衰期，t1/2）。通过差示扫描量热法（DSC）和圆二色光谱法（CD）测定酶的热变性。以α-淀粉酶（5A2A）为模板对AGXA进行同源建模。
有钙离子时，AGXA的Topt、T50、t1/2、Tm和ΔH值显著高于无钙离子的AGXA，表明钙离子对α-淀粉酶结构具有稳定作用，且随温度升高而增强。基于DSC测量，AGXA通过两态不可逆展开过程发生热变性。基于CD光谱，无钙离子的AGXA在展开时呈现两个转变状态，包括α螺旋含量增加以及从α螺旋向β折叠结构的转变，这与有Ca2+离子的AGXA明显不同，根据建模结构，多达4个Ca2+离子位于结构域间或结构域内区域。
这些结果表明Ca2+离子对AGXA结构的热稳定性有显著影响。
