Abstract Monellin, one kind of most sweet proteins, could be used instead of carbohydrates in the diabetic diet and pharmaceuticals. However, the critical problem interferes with monellin usage in the food or pharmaceutical industry was its unstable at high temperatures. Here, we describe a novel method to increase the thermostability of monellin. Inspired by the high Tm value in RNA hairpin containing structure, the beta-hairpin was added between the two polypeptide chains of monellin instead of Gly-Phe dipeptide linker. Three kinds of beta-hairpin libraries were constructed and selected. Remarkably, the Tm has been increased from 54.7℃ of monellin with Gly-Phe dipeptide linker to 79.5℃, 89.1℃ and 89.4℃ of selected three mutants. And combined with E24Q/Y80R mutation, the mutated E24Q/Y80R hairpin monellin Tm was as high as 96.1℃. It was soluble even heated in boiling water for 10 min, and sweetness recovered after cooling. This new hairpin monellin may show remarkable potential for further sweeteners.

中文翻译：

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一种基于莫内林的高温稳定甜蛋白的设计与开发

摘要 莫内林是一种最甜的蛋白质，可替代碳水化合物用于糖尿病饮食和药物治疗。然而，影响莫内林在食品或制药工业中使用的关键问题是其在高温下不稳定。在这里，我们描述了一种提高莫内林热稳定性的新方法。受含有 RNA 发夹结构的高 Tm 值的启发，在莫内林的两条多肽链之间添加了 β-发夹，而不是 Gly-Phe 二肽接头。构建并选择了三种β-发夹文库。值得注意的是，Tm 从带有 Gly-Phe 二肽接头的 monellin 的 54.7℃ 增加到选定的三个突变体的 79.5℃、89.1℃ 和 89.4℃。并结合E24Q/Y80R突变，突变的E24Q/Y80R发夹monellin Tm高达96.1℃。在沸水中加热10分钟即可溶解，冷却后恢复甜味。这种新的发夹莫内林可能显示出作为进一步甜味剂的显着潜力。