Thermodynamic stability of a cold-active α-amylase from the Antarctic bacterium Alteromonas haloplanctis
Thermodynamic stability of a cold-active α-amylase from the Antarctic bacterium Alteromonas haloplanctis
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DOI：
10.1021/bi982650
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发表时间：
1999-04-06
期刊：
影响因子：
2.9
通讯作者：
Gerday, C
中科院分区：
文献类型：
--
作者：
Feller, G;d'Amico, D;Gerday, C
利用内源荧光、圆二色谱和差示扫描量热法研究了南极细菌Alteromonashaloplanctis分泌的低温α-淀粉酶（AHA）的热稳定性。结果发现，这种热不稳定的酶是最大的已知的多结构域蛋白表现出可逆的两种状态的展开，证明了连续的量热转换后的恢复Δ H-cal值，Δ H-cal/Δ H-eff比接近统一，和展开的热力学参数的扫描速率的独立性。相比之下，嗜中温α-淀粉酶研究（从猪胰腺，人类唾液腺，黄粉虫，芽孢杆菌amyloliquefaciens，和地衣芽孢杆菌）不可逆地展开根据非两态机制，不像嗜中温α-淀粉酶，AHA的熔点是独立的钙和氯离子结合，而这些离子的变构和结构功能是保守的。AHA在最佳条件下的热稳定性的特征在于T-m为43.7 ℃，Δ H-cal为238 kcal mol（-1），Δ C-p为8.47 kcal mol（-1）K-1。这些值用于计算在宽温度范围内展开的吉布斯自由能。该稳定性曲线显示：（a）AHA [22卡]的比Delta G（最大值）（mol残基）（-1）]比嗜中温α-淀粉酶低4倍，（B）基团水合作用在低温下酶的柔性中起关键作用，（c）冷解折叠的温度与细菌生长的下限密切对应，和（d）重组热不稳定酶可在中等温度下在嗜温宿主中表达。也有人认为，冷活性α-淀粉酶已经朝着其天然状态的最低可能的构象稳定性发展。
The thermal stability of the cold-active alpha-amylase (AHA) secreted by the Antarctic bacterium Alteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and differential scanning calorimetry. It was found that this heat-labile enzyme is the largest known multidomain protein exhibiting a reversible two-state unfolding, as demonstrated by the recovery of Delta H-cal values after consecutive calorimetric transitions, a Delta H-cal/Delta H-eff ratio close to unity, and the independence of unfolding thermodynamic parameters of scan rates. By contrast, the mesophilic alpha-amylases investigated here (from porcine pancreas, human salivary glands, yellow meal beetle, Bacillus amyloliquefaciens, and Bacillus licheniformis) unfold irreversibly according to a non-two-state mechanism, Unlike mesophilic alpha-amylases, the melting point of AHA is independent of calcium and chloride binding while the allosteric and structural functions of these ions are conserved. The thermostability of AHA at optimal conditions is characterized by a T-m of 43.7 degrees C, a Delta H-cal of 238 kcal mol(-1), and a Delta C-p of 8.47 kcal mol(-1) K-1. These values were used to calculate the Gibbs free energy of unfolding over a wide range of temperatures. This stability curve shows that (a) the specific Delta G(max) of AHA [22 cal (mol of residue)(-1)] is 4 times lower than that of mesophilic alpha-amylases, (b) group hydration plays a crucial role in the enzyme flexibility at low temperatures, (c) the temperature of cold unfolding closely corresponds to the lower limit of bacterial growth, and (d) the recombinant heat-labile enzyme can be expressed in mesophilic hosts at moderate temperatures. It is also argued that the cold-active alpha-amylase has evolved toward the lowest possible conformational stability of its native state.