Simultaneous boosting of thermostability and glucose tolerance in engineered glucosidase through terminal...

Rare ginsenosides exhibit significant market potential owing to their superior pharmacological activity and bioavailability. However, their large-scale green production remains challenging due to the poor thermostability and glucose tolerance of natural enzymes. In this study, we propose a rational terminal engineering strategy to simultaneously enhance the thermostability and glucose tolerance of β-glucosidase, a key enzyme in ginsenoside bioconversion. Through terminal flexibility analysis, we developed an N-terminally truncated variant (GlSK 20aa), which demonstrated a 9.17-fold extended half-life at 40 °C and a 5.2-fold improvement in glucose tolerance compared to the wild-type enzyme. Mechanistic studies revealed that terminal truncation induces structural rigidification, resulting in a significant increase in thermostability. Meanwhile, the exposure of charged residues on the protein surface effectively redirects glucose binding from the catalytic pocket to the enzyme surface, thereby reducing competitive inhibition. The engineered enzyme exhibited exceptional catalytic efficiency in the high-titer synthesis of ginsenoside F1, achieving a 4.03-fold higher yield under high substrate concentrations. Our findings uncover the importance of flexible terminal regions in enzyme stability and demonstrate that terminal truncation serves as a simple, powerful strategy to optimize biocatalysis for industrial processes involving high temperatures and sugar-rich byproducts.