Thermal Stability Enhancement of a Carbohydrate-Binding Module for Hyaluronic Acid Based on an Integrated Computational Screening Strategy

Hyaluronic acid (HA) is widely used in the food, pharmaceutical, and cosmetic industries, where its content directly determines product efficacy. Therefore, industrial production urgently requires precise and robust quantitative analytical methods. While rapid detection methods for polysaccharides based on carbohydrate-binding modules (CBMs) have been established, the limited thermal stability of most natural CBMs restricts their application. This study employed a comprehensive rational design strategy to further enhance the thermal stability of SrCBM70, which is a previously identified HA-specific binding protein. Compared with the wild-type, 83% of first-round mutants exhibited elevated melting temperatures (Tm), while 100% of second-round mutants demonstrated improvement. The optimal mutant, A19W/K38R, achieved a Tm of 69.32 °C. Structural analysis attributed improvements to strengthened hydrophobic cores and hydrogen-bond networks. This study demonstrates the potential for modifying the thermal stability of the CBM and the feasibility of rational design strategies to enhance its stability.