Abstract
Enhancing the thermostability of glucose oxidase (GOD) is crucial for food science applications. However, traditional design methods based on a single GOD structure are ineffective especially in extreme thermal environments. 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. The residual activity of V402F was six times that of M8 at 80 ℃ for 2 min, and enzyme activity was even slightly increased. 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 GOD variant with improved thermostability, as well as a more precise and efficient design strategy for GOD and other flexible enzymes.