State Key Lab of Microbial Resources, Institute of Microbiology, Chinese Academy of Science, Beijing 100101, China.
J Biosci Bioeng. 2010 Jul;110(1):12-7. doi: 10.1016/j.jbiosc.2009.12.002. Epub 2010 Jan 3.
Thermostability can be increased by introducing prolines at suitable sites in target proteins. In this study, we compared five thermostable alpha-glucosidases and the moderate thermostable alpha-glucosidase (TtGluA) from Thermoanaerobacter tengcongensis MB4. Based on the amino acid sequence alignment, four sites (Leu152, Asn208, Lys285, and Thr430) of TtGluA were chosen for proline substitution to improve its thermostability. Thermostability of mutants L152P, K285P, and T430P increased evidently, but no thermostability improvement was observed for N208P. Compared to the wild-type enzyme, T(50)(15) of T430P had a rise of 2 degrees C without distinct loss of activity. However, T(50)(15) values of L152P and K285P increased 2 degrees C and 10.5 degrees C, respectively, while retaining activity of only 26.6% and 24.9% of wild-type enzyme. The K(m) of L152P, K285P, T430P and wild-type enzyme was 1.61, 0.32, 1.64, and 1.08 mM, respectively. These indicate that the selected sites are not only important for the thermostability but also related to the substrate binding and catalytic activity of TtGluA. The CD spectra analysis of the improved mutants and wild-type enzyme showed no distinct changes in their secondary structures. Combining analysis of secondary structure prediction and 3D structure modeling, the proline substitution at the three sites stabilized TtGluA possibly by reducing the flexibility of loop and random coil or (and) increasing the hydrophobic effect at these strategic regions with no evident structure change.
通过在靶蛋白的合适位置引入脯氨酸，可以提高其热稳定性。在这项研究中，我们比较了五种耐热α-葡萄糖苷酶和来自嗜热厌氧菌 MB4 的中温耐热α-葡萄糖苷酶（TtGluA）。基于氨基酸序列比对，选择了 TtGluA 的四个位点（Leu152、Asn208、Lys285 和 Thr430）进行脯氨酸取代，以提高其热稳定性。突变体 L152P、K285P 和 T430P 的热稳定性明显提高，但 N208P 没有观察到热稳定性提高。与野生型酶相比，T430P 的 T(50)(15)升高了 2°C，而活性没有明显损失。然而，L152P 和 K285P 的 T(50)(15)值分别升高了 2°C 和 10.5°C，而保留的活性仅为野生型酶的 26.6%和 24.9%。L152P、K285P、T430P 和野生型酶的 K(m)分别为 1.61、0.32、1.64 和 1.08 mM。这表明所选的位点不仅对热稳定性很重要，而且与 TtGluA 的底物结合和催化活性有关。对改良突变体和野生型酶的 CD 光谱分析表明，它们的二级结构没有明显变化。结合二级结构预测和 3D 结构建模分析，三个位点的脯氨酸取代可能通过降低环和无规卷曲的灵活性或（和）增加这些关键区域的疏水性效应来稳定 TtGluA，而没有明显的结构变化。
