Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
J Microbiol Biotechnol. 2012 May;22(5):592-9. doi: 10.4014/jmb.1109.09009.
Bacillus licheniformis alpha-amylase (BLA), a thermophilic counterpart of Bacillus amyloliquefaciens alpha-amylase (BAA), is an appropriate model for the design of stabilizing mutations in BAA. BLA has 10 more histidines than BAA. Considering this prominent difference, in the present study, three out of these positions (I34, Q67, and P407; located in the thermostability determinant 1 region and Ca-III binding site of BAA) were replaced with histidine in BAA, using the site-directed mutagenesis technique. The results showed that the thermostability of P407H and Q67H mutants had increased, but no significant changes were observed in their kinetic parameters compared to that of the wild type. I34H replacement resulted in complete loss of enzyme activity. Moreover, fluorescence and circular dichroism data indicated a more rigid structure for the P407H variant compared with that of the wild-type BAA. However, the flexibility of Q67H and I34H mutants increased in comparison with that of wild-type enzyme.
地衣芽孢杆菌α-淀粉酶（BLA）是解淀粉芽孢杆菌α-淀粉酶（BAA）的耐热对应物，是设计 BAA 稳定突变的合适模型。BLA 比 BAA 多 10 个组氨酸。考虑到这一显著差异，在本研究中，使用定点突变技术，将 BAA 中的三个位置（位于耐热性决定簇 1 区域和 Ca-III 结合位点的 I34、Q67 和 P407）的组氨酸替换为组氨酸。结果表明，P407H 和 Q67H 突变体的热稳定性增加，但与野生型相比，其动力学参数没有明显变化。I34H 取代导致酶完全失活。此外，荧光和圆二色性数据表明，与野生型 BAA 相比，P407H 变体的结构更具刚性。然而，与野生型酶相比，Q67H 和 I34H 突变体的灵活性增加。
