Design and development of a high temperature stable sweet protein base on monellin

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.