差示扫描量热法分析Ca^2+对嗜热菌来源的α-淀粉酶热稳定性的影响  

Effect of Calcium Ions on the Thermostability of α-Amylase from Thermophiles Analyzed by DSC

作　　者：廖思明[1,2] 梁戈 申乃坤 卢波[2] 芦志龙[1,2] 彭立新 黄纪民[2] 周锋[2] 韦宇拓[1] 黄日波[1,2] Liao Siming;Liang Ge;Shen Naikun;Lu Bo;Lu Zhilong;Peng Lixin;Huang Jimin;Zhou Feng;Wei Yutuo;Huang Ribo(College of Life Science and Technology,Guangxi University,Nanning,530005;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,Nanning,530007;School of Marine Sciences and Biotechnology,Guangxi Key Laboratory of Utilization of Microbial and Botanical Resources,Guangxi University for Nationalities,Nanning,530008)

机构地区：[1]广西大学生命科学与技术学院,南宁530005 [2]广西科学院,国家非粮生物质能源工程技术研究中心,非粮生物质酶解国家重点实验室,广西生物炼制重点实验室,南宁530007 [3]广西民族大学海洋与生物技术学院,广西高校微生物与植物资源利用重点实验室,南宁530008

出　　处：《基因组学与应用生物学》2020年第4期1655-1661,共7页Genomics and Applied Biology

基　　金：国家自然科学基金(31560251);广西自然科学基金(2015GXNSFAA139053);广西科技计划(桂科合151040-01-6)项目共同资助。

摘　　要：采用毛细管差示扫描量热法,分析Ca^2+对嗜热菌Anoxybacillus sp.来源的α-淀粉酶AGXA的热稳定性的影响及其机制。脱气后的蛋白样品和缓冲液,分别加入对应的样品池和缓冲液池中,测量温度为10℃~120℃,升温速率为1℃/min。测量结果中,纵坐标为Cp,横坐标为温度,去折叠变化曲线峰最高点对应的横坐标为蛋白质去折叠温度Tm,去折叠变化曲线与对应温度的积分值为热焓变化值ΔH,范特霍夫焓变化值ΔHv由去折叠变化曲线峰形状决定。根据改进的吉布斯-亥姆霍兹方程计算AGXA的自由能变化值ΔG,绘制AGXA自由能变化与温度关系的稳定性曲线。结果表明:有Ca^2+和无Ca^2+存在时,AGXA的ΔHv与ΔH的比值都约等于1.0;有Ca^2+时,AGXA的Tm、ΔH和ΔCp值,分别为77.8℃、292.5 kcal/mol和2.76 kcal·mol^-1·℃^-1;无Ca^2+时,AGXA的Tm、ΔH和ΔCp值,则分别为67.3℃、238.3 kcal/mol和3.81 kcal·mol^-1·℃^-1;有Ca^2+时的AGXA,其Tm、ΔH值分别比无Ca^2+时的高,ΔCp值则比无Ca^2+时的低。有Ca^2+和无Ca^2+的α-淀粉酶AGXA的热变性过程皆为不可逆的双态模式,有Ca^2+的AGXA,通过增大ΔH和降低ΔCp的方式提高其热稳定性。研究揭示了Ca^2+提高AGXA的热稳定性机制,为进一步扩大该酶的应用提供了理论和实践支撑。The effect and mechanism of calcium ions on the thermostability ofα-amylase from thermophilic Anoxybacillus sp.(AGXA)was determined by capillary differential scanning calorimeter.After degassed,the protein sample and buffer were loaded into the sample and reference cells,respectively.And the measurements were performed by increasing the temperature from 10℃to 120℃at 1℃/min scan rate.Protein sample scans were corrected by subtraction of the respective buffer from the baseline values.The apparent Tm was determined as the temperature corresponding to maximum Cp,the calorimetric enthalpyΔH corresponded to the area under the peak of the heat capacity versus temperature graph,and the van't Hoff enthalpyΔHv was determined by the shape of the transition peak.The free energy changeΔG data of AGXA was determined according to a modified version of the Gibbs-Helmholtz equation.And the protein stability curve,showing plots of free energy of stabilization as a function of temperature,was described also.The results showed that the ratio of calorimetric heat change(ΔH)to van't Hoff heat change(ΔHv)in the transition curves was approximately equal to 1.0,indicating theα-amylases with Ca^2+and without Ca^2+were two-state irreversible.In the presence of Ca^2+,the thermal denaturation temperature,calorimetric enthalpy change and heat capacity change were 77.8℃,292.5 kcal/mol and 2.76 kcal·mol^-1·℃^-1,respectively,which were 10.5℃higher,53.1 kcal/mol higher and 1.05 kcal·mol^-1·℃^-1 lower respectively,than those observed in theα-amylase lacking Ca^2+.AGXA underwent thermal denaturation by adopting two-state irreversible unfolding processes.With Ca^2+,the stability of AGXA was improved by the way of increasing the calorimetric enthalpy change and decreasing the heat capacity change.These studies revealed the mechanism of how Ca^2+binding increase thermostability ofα-amylase from thermophiles,which providing theoretical and practical support for further expanding the application of the enzyme.

关 键 词：Α-淀粉酶 钙离子 热稳定性 差示扫描量热法 

分 类 号：Q936[生物学—微生物学]