Structural stability of soybean (Glycine max) α-amylase: properties of the unfolding transition studied with fluorescence and CD spectroscopy.
Structural stability of soybean (Glycine max) α-amylase: properties of the unfolding transition studied with fluorescence and CD spectroscopy.
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大豆 (Glycine max) α-淀粉酶的结构稳定性：用荧光和 CD 光谱研究解折叠转变的特性。
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发表时间：
2011
期刊：
影响因子：
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通讯作者：
A. Kayastha
中科院分区：
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作者：
A. Kumari;T. Rosenkranz;J. Fitter;A. Kayastha
哺乳动物和微生物α-淀粉酶的稳定性和去折叠已被深入研究。然而，关于植物α-淀粉酶的结构稳定性，即来自大麦的两种同工酶AMY 1和AMY 2、来自绿豆（Vigna radiata）的α-淀粉酶和来自麦芽高粱（Sorcopbicolor）的α-淀粉酶的结构稳定性，仅有有限的信息。本文采用圆二色谱法和荧光光谱法研究了大豆α-淀粉酶（GMA）对高温和化学变性剂（盐酸胍）的稳定性。由于众所周知钙离子对α-淀粉酶的酶活性和稳定性起着至关重要的作用，我们用钙结合和钙游离的GMA进行了研究。热去折叠转变温度从钙饱和样品的72°C降低到钙耗尽的GMA的情况下的57°C。类似地，GndHCl转变浓度从钙结合GMA的0.70 M降低至不存在钙时的0.41 M。由于未折叠状态的聚集，GMA的热未折叠不可逆。在含有7 M甘油的天然缓冲液中稀释未折叠的酶后，在6 M GndHCl中未折叠的GMA显示出高度的可逆性。此外，复性的酶显示93%的活性。
Stability and unfolding of mammalian and microbial α-amylases have been intensively investigated. However, there is only limited information available on the structural stability of plant α-amylases, namely of the two isoenzymes from barley AMY1 and AMY2, of the α-amylase from mung bean (Vigna radiata), and of the α-amylase from malted sorghum (Sorghum bicolor). We report here the stability of soyabean α-amylase (GMA), against elevated temperatures and chemical denaturants (GndHCl) by employing circular dichroism and fluorescence spectroscopy. Since it is well-known that calcium ions play a crucial role for enzymatic activity and stability of a-amylases, we performed our studies with calcium bound and calcium free GMA. The thermal unfolding transition temperature decreased from 72°C for calcium saturated samples to 57°C for the case of calcium depleted GMA. Similarly, the GndHCl transition concentration was lowered from 0.70 M for calcium bound GMA to 0.41 M in the absence of calcium. Thermal unfolding of GMA irreversible due to aggregation of the unfolded state. GMA unfolded in 6 M GndHCl shows high degree of reversibility after diluting the unfolded enzyme in native buffer containing 7 M glycerol. Furthermore, the refolded enzyme showed 93% of activity.