Laboratoire d'Enzymes et de Métabolites des Procaryotes, Centre de Biotechnologie de Sfax, Université de Sfax, BP "1177", 3018 Sfax, Tunisia.
J Biotechnol. 2013 Dec;168(4):601-6. doi: 10.1016/j.jbiotec.2013.08.026. Epub 2013 Aug 29.
The thermostability of maltogenic amylase from Bacillus sp. US149 (MAUS149) was improved by random mutagenesis using error prone PCR. The library constructed for the mutants obtained was subjected to screening, leading to the selection of a thermostable mutant enzyme named MA-A27. The latter was noted to contain four single mutations, namely D46V, P78L, V145A, and K548E. The half-life times recorded for MA-A27 at 50°C and 55°C were 70 min and 25 min, compared to 30 min and 13 min for the wild type, respectively. The results from molecular modeling attributed the increase in thermostability observed for MA-A27 to P78L and K548E substitutions that led to new hydrogen bond and salt bridge formations. Further site-directed mutagenesis studies showed that the P78L and K548E single mutations underwent an increase in thermostability, thus confirming the joint contribution of both substitutions to the increase in thermostability observed for MA-A27.
通过易错 PCR 对来自芽孢杆菌的麦芽寡糖基淀粉酶（MAUS149）进行随机诱变以提高其热稳定性。对获得的突变体文库进行筛选，从而选择了一种名为 MA-A27 的热稳定突变酶。后者被发现含有四个单点突变，即 D46V、P78L、V145A 和 K548E。与野生型相比，MA-A27 在 50°C 和 55°C 下的半衰期分别为 70 分钟和 25 分钟，而野生型分别为 30 分钟和 13 分钟。分子建模的结果表明，观察到的 MA-A27 热稳定性增加归因于 P78L 和 K548E 取代导致形成新的氢键和盐桥。进一步的定点突变研究表明，P78L 和 K548E 的单点突变增加了热稳定性，从而证实了这两个取代对 MA-A27 观察到的热稳定性增加的共同贡献。
