Nielsen Anders D, Pusey Marc L, Fuglsang Claus C, Westh Peter
Department of Life Sciences and Chemistry, Roskilde University, P.O. Box 260, 1-Universitetsvej, DK-4000 Roskilde, Denmark.
Biochim Biophys Acta. 2003 Nov 3;1652(1):52-63. doi: 10.1016/j.bbapap.2003.08.002.
The thermal stability of a recombinant alpha-amylase from Bacillus halmapalus alpha-amylase (BHA) has been investigated using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). This alpha-amylase is homologous to other Bacillus alpha-amylases where crystallographic studies have identified the existence of three calcium binding sites in the structure. Denaturation of BHA is irreversible with a T(m) of approximately 89 degrees C and DSC thermograms can be described using a one-step irreversible model. A 5 degrees C increase in T(m) in the presence of 10-fold excess CaCl(2) was observed. However, a concomitant increase in the tendency to aggregate was also observed. The presence of 30-40-fold excess calcium chelator (ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis[beta-aminoethyl ether] N,N,N',N'-tetraacetic acid (EGTA)) results in a large destabilization of BHA, corresponding to about 40 degrees C lower T(m) as determined by both CD and DSC. Ten-fold excess EGTA reveals complex DSC thermograms corresponding to both reversible and irreversible transitions, which probably originate from different populations of BHA/calcium complexes. Combined interpretation of these observations and structural information on homologous alpha-amylases forms the basis for a suggested mechanism underlying the inactivation mechanism of BHA. The mechanism includes irreversible thermal denaturation of different BHA/calcium complexes and the calcium binding equilibria. Furthermore, the model accounts for a temperature-induced reversible structural change associated with calcium binding.
利用圆二色光谱法（CD）和差示扫描量热法（DSC）对来自嗜盐芽孢杆菌的重组α-淀粉酶（BHA）的热稳定性进行了研究。这种α-淀粉酶与其他芽孢杆菌α-淀粉酶同源，晶体学研究已确定其结构中存在三个钙结合位点。BHA的变性是不可逆的，其熔点（T(m)）约为89℃，DSC热谱图可用一步不可逆模型描述。在存在10倍过量CaCl₂的情况下，观察到T(m)升高了5℃。然而，同时也观察到聚集倾向增加。存在30 - 40倍过量的钙螯合剂（乙二胺四乙酸（EDTA）或乙二醇双[β-氨基乙醚]N,N,N',N'-四乙酸（EGTA））会导致BHA大幅失稳，通过CD和DSC测定，其T(m)降低约40℃。10倍过量的EGTA显示出对应可逆和不可逆转变的复杂DSC热谱图，这可能源于不同群体的BHA/钙复合物。对这些观察结果与同源α-淀粉酶结构信息的综合解读，构成了BHA失活机制潜在机制的建议基础。该机制包括不同BHA/钙复合物的不可逆热变性以及钙结合平衡。此外，该模型解释了与钙结合相关的温度诱导可逆结构变化。
