State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China; International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, Jiangsu 214122, China.
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China; International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, Jiangsu 214122, China.
Int J Biol Macromol. 2023 Jul 1;242(Pt 2):124871. doi: 10.1016/j.ijbiomac.2023.124871. Epub 2023 May 16.
Alginate lyases with strict substrate specificity possess potential in directed production of alginate oligosaccharides with specific composition. However, their poor thermostability hampered their applications in industry. In this study, an efficient comprehensive strategy including sequence-based analysis, structure-based analysis, and computer-aid ΔΔG value calculation was proposed. It was successfully performed on alginate lyase (PMD) with strict poly-β-D-mannuronic acid substrate specificity. Four single-point variants A74V, G75V, A240V, and D250G with increased T of 3.94 °C, 5.21 °C, 2.56 °C, and 4.80 °C, respectively, were selected out. After ordered combined mutations, a four-point mutant (M4) was finally generated which displayed remarkable increase on thermostability. The T of M4 increased from 42.25 °C to 51.59 °C and its half-life at 50 °C was about 58.9-fold of PMD. Meanwhile, there was no obvious loss of enzyme activity (more than 90% retained). Molecular dynamics simulation analysis insisted that the improvement of thermostability might be attribute to the rigidified region A which might be caused by the newly formed hydrogen bonds and salt bridges introduced by mutations, the lower distance of original hydrogen bonds, and the more compact overall structures.
具有严格底物特异性的褐藻胶裂解酶在定向生产具有特定组成的褐藻寡糖方面具有潜力。然而，它们较差的热稳定性限制了它们在工业中的应用。在这项研究中，提出了一种包括基于序列分析、基于结构分析和计算机辅助ΔΔG 值计算的高效综合策略。该策略成功应用于具有严格聚-β-D-甘露糖酸底物特异性的褐藻胶裂解酶（PMD）。分别选择了四个单点突变体 A74V、G75V、A240V 和 D250G，它们的 T 分别提高了 3.94°C、5.21°C、2.56°C 和 4.80°C。经过有序的组合突变，最终生成了一个四点突变体（M4），其热稳定性显著提高。M4 的 T 从 42.25°C 增加到 51.59°C，其在 50°C 下的半衰期是 PMD 的约 58.9 倍。同时，酶活性没有明显损失（保留超过 90%）。分子动力学模拟分析表明，热稳定性的提高可能归因于刚性区域 A，这可能是由突变引入的新形成的氢键和盐桥、原始氢键的更小距离以及更紧凑的整体结构引起的。
