Declerck N, Joyet P, Trosset J Y, Garnier J, Gaillardin C
Laboratoire de Génétique, Centre des Biotechnologies Agro-Industtrielles, INAP-G, Thiverval-Grignon, France.
Protein Eng. 1995 Oct;8(10):1029-37. doi: 10.1093/protein/8.10.1029.
We have identified previously two critical positions for the thermostability of the highly thermostable alpha-amylase from Bacillus licheniformis. We have now introduced all 19 possible amino acid residues to these two positions, His133 and Ala209. The most favourable substitutions were to Ile and Val, respectively, which both increased the half-life of the enzyme at 80 degrees C by a factor of approximately 3. At both positions a stabilizing effect of hydrophobic residues was observed, although only in the case of position 133 could a clear correlation be drawn between the hydrophobicity of the inserted amino acid and the gain in protein stability. The construction of double mutants showed a cumulative effect of the most favourable and/or deleterious substitutions. Computer modelling was used to generate a 3-D structure of the wild-type protein and to model substitutions at position 209, which lies in the conserved (alpha/beta)8 barrel domain of alpha-amylase; Ala209 would be located at the beginning of the third helix of the barrel, in the bottom of a small cavity facing the fourth helix. The model suggests that replacement by, for example, a valine could fill this cavity and therefore increase intra- and interhelical compactness and hydrophobic interactions.
我们之前已经确定了地衣芽孢杆菌中高度耐热的α-淀粉酶热稳定性的两个关键位置。现在，我们将所有19种可能的氨基酸残基引入到这两个位置，即His133和Ala209。最有利的取代分别是异亮氨酸和缬氨酸，这两种取代都使该酶在80℃下的半衰期增加了约3倍。在这两个位置都观察到了疏水残基的稳定作用，尽管只有在位置133的情况下，才能在插入氨基酸的疏水性与蛋白质稳定性的增加之间建立明确的相关性。双突变体的构建显示出最有利和/或有害取代的累积效应。计算机建模用于生成野生型蛋白质的三维结构，并对位于α-淀粉酶保守的(α/β)8桶状结构域中的位置209处的取代进行建模；Ala209位于桶状结构第三个螺旋的起始处，在面对第四个螺旋的一个小腔的底部。该模型表明，例如用缬氨酸取代可以填充这个腔，从而增加螺旋内和螺旋间的紧密性以及疏水相互作用。
