Complementary Distant and Active Site Mutations Simultaneously Enhance Catalytic Activity and Thermostability of α-Galactosidase

The industrial applications of enzymes are limited due to the activity-stability trade-off, which implies that the improvement of thermostability often accompanies decreased activity. This study presents a dual-strategy approach to simultaneously improve the catalytic efficiency and thermostability of α-galactosidase galV from <i>Anoxybacillus vitaminiphilus</i> WMF1. Our integrated method combines computational analysis with enzyme property prediction to selectively target and modify the catalytic region and residues that are distant from the active site. We identified and experimentally validated mutations that improve activity without compromising stability and further increased thermostability through additional distant-site mutations. The resulting mutant enzyme variant N549Q/T550N/Y634F demonstrated a 6.2-fold increase in catalytic efficiency and a 3.2-fold improvement in the half-life at 65 °C. Molecular dynamics (MD) simulations supported the structural basis for the observed enhancements. This approach offers a refined strategy for engineering α-galactosidases with improved industrial applicability, overcoming the traditional trade-offs between enzyme activity and stability. Hydrolytic activity toward raffinose family oligosaccharides (RFOs) was validated using soymilk as a model substrate, demonstrating significant practical potential.