A series of mutations (N130D, G260A, S437T, N130D/G260Q, N130D/S437Y) showed higher thermostability and activity than the wild-type enzyme. Thus, N130D/G260Q exhibited a 6.6-fold increase in half-life and 2.45 °C increase in unfolding temperature; N130D/S437Y showed a 10 °C increase in optimal temperature. The secondary structure did not change much that contributed to improved thermostability were investigated in detail using circular dichroism. Homology modeling suggested that enhanced thermostability and specific activity may result from favorable hydrophobic interactions.
A series of mutations were achieved, showing higher thermostability and activity than the wild-type enzyme by semi-rational mutagenesis with limited structure information. Our findings provide important new insights into molecular modifications aimed at improving Ana-rLOX thermostability and activity.
一系列突变(N130D,G260A,S437T,N130D/G260Q,N130D/S437Y)显示出比野生型酶更高的热稳定性和活性。因此,N130D/G260Q的半衰期增加了6.6倍,展开温度增加了2.45°C;N130D/S437Y的最佳温度增加了10°C。二级结构变化不大,这有助于提高热稳定性,使用圆二色性进行了详细研究。同源性建模表明,增强的热稳定性和比活性可能是有利的疏水相互作用的结果。
实现了一系列突变,通过具有有限结构信息的半理性诱变显示出比野生型酶更高的热稳定性和活性。我们的发现为旨在改善Ana-rLOX热稳定性和活性的分子修饰提供了重要的新见解。