College of Food Science and Light Industry, Nanjing Tech University, Nanjing 211816, China; College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China.
College of Food Science and Light Industry, Nanjing Tech University, Nanjing 211816, China.
Int J Biol Macromol. 2021 Jan 31;168:13-21. doi: 10.1016/j.ijbiomac.2020.11.195. Epub 2020 Dec 4.
One of the most desirable properties for industrial enzymes is high thermotolerance, which can reduce the amount of biocatalyst used and lower the production cost. Aiming to improve the thermotolerance of trehalose synthase (TreS, EC 5.4.99.16) from Thermomonospora curvata, four mutants (G78D, V289L, G322A, I323L) and four cyclized TreS variants fused using different Tag/Catcher pairs (SpyTag-TreS-SpyCatcher, SpyTag-TreS-KTag, SnoopTag-TreS-SnoopCatcher, SnoopTagJR-TreS-DogTag) were constructed. The results showed that cyclization led to a much larger increase of thermostability than that achieved via site-directed mutagenesis. The t of all four cyclized TreS variants at 55 °C increased 2- to 3- fold, while the analysis of kinetic and thermodynamic stability indicated that the T of the different cyclized TreS variants increased by between 7.5 °C and 15.5 °C. Molecular dynamics simulations showed that the Rg values of cyclized TreS decreased significantly, indicating that the protein maintained a tight tertiary structure at high temperatures, avoiding exposure of the hydrophobic core to the solvent. Cyclization using a Tag/Catcher pair is a simple and effective method for improving the thermotolerance of enzymes.
工业酶的一个最理想的特性是高热稳定性，这可以减少生物催化剂的用量并降低生产成本。本研究旨在提高来自嗜热栖热菌的海藻糖合酶（TreS，EC 5.4.99.16）的耐热性，构建了四个突变体（G78D、V289L、G322A、I323L）和四个使用不同 Tag/Catcher 对融合的环化 TreS 变体（SpyTag-TreS-SpyCatcher、SpyTag-TreS-KTag、SnoopTag-TreS-SnoopCatcher、SnoopTagJR-TreS-DogTag）。结果表明，环化导致的热稳定性增加比定点突变大得多。所有四个环化 TreS 变体在 55°C 时的 t 值增加了 2-3 倍，而动力学和热力学稳定性分析表明，不同环化 TreS 变体的 T 值增加了 7.5°C 到 15.5°C。分子动力学模拟表明，环化 TreS 的 Rg 值显著降低，表明该蛋白质在高温下保持紧密的三级结构，避免疏水性核心暴露于溶剂中。使用 Tag/Catcher 对的环化是提高酶耐热性的简单有效方法。
