Enhancing the Stability of Sweet Protein Neoculin for Food Applications Using Computational Strategies

Sweet proteins, known for their high sweetness and low caloric content, offer great potential as safe, low-calorie sweeteners without the negative health effects associated with sugar or artificial alternatives. Neoculin, unique for its dual properties of sweetness and taste-modifying, holds significant promise for food industry applications. However, its low thermal stability limits its broader use in food processing, highlighting the need to improve its stability. In this study, we applied disulfide bonds design and saturation mutagenesis based on the PyRosetta algorithm to improve neoculin's thermal stability. Experimental screening revealed that introducing a pair of disulfide bonds increased the melting temperature (<i>T</i>_m) by 4.7 °C, and subsequent saturation mutagenesis led to a mutant with a significant <i>T</i>_m increase of 14.5 °C. This combined approach resulted in a final mutant (FM) with a <i>T</i>_m of 84.7, 20.3 °C higher than the wild-type (WT), while maintaining sweetness. Furthermore, Gaussian network model (GNM) analysis indicated that the cross-correlation of specific residues in FM was enhanced compared to WT, which may explain the improved thermal stability. This study presents a neoculin variant with substantial industrial application potential in the food and beverage sectors and offers valuable strategies for future protein engineering of low-calorie sweeteners.