Enhancing the Thermostability of Sweet Protein Thaumatin II by Integrated Rational Design

Abstract Thaumatin II is a high-intensity sweet protein whose limited thermostability restricts its industrial application. Using the previously evolved high-yield Thaumatin variant M-882 as the parental scaffold, we used an integrated rational design strategy combining HotSpot Wizard, FoldX, and PyRosetta. Candidate variants were evaluated by sfGFP-based fluorescence screening, SDS-PAGE, and absolute protein quantification. The variant R178H showed the most favorable overall balance of structural compatibility and thermal resilience. After heating at 80 °C for 2 h, R178H retained 63% of its soluble protein fraction, compared with 43% for M-882. Structural analysis suggested that replacing Arg178 with histidine reduced local steric hindrance and improved packing interactions. Molecular dynamics simulations further indicated that R178H adopted a more compact and stable conformation. These results clarify the structural basis for its improved heat resistance and provide a rational strategy for engineering thermostable sweet proteins for food-processing applications.