Break through the thermostability of glucose oxidase in extremely thermal environments with a novel dynamic ensemble design protocol

Enhancing the thermostability of glucose oxidase (Gox) is crucial for its industrial applications. However, in traditional design methods based on a single Gox structure, hundreds of or several rounds of variants were predicted and tested, with limited thermostability enhancement under high temperature conditions. Here, we established a method for precisely locating residue by analysing the dynamic conformations of GoxM8 (M8) and further enhancing thermostability while maintaining activity. Our novel dynamic ensemble approach, coupled with FireProt computational analyses, was used to obtain the best mutant, V402F, from diverse conformations of M8. V402F residual activity was six times that of M8 at 80 ℃ for 2 min, with no loss of enzyme activity. Experimental validation and computational analysis of stability mechanisms demonstrated the deficiencies of previous design strategies for flexible enzymes, proving the validity of our approach. Thus, we present a Gox variant with improved thermostability, as well as a more precise and efficient design strategy for Gox and other flexible enzymes.