Semi-rational design of a hyperstable β-glucosidase Bgl1269 for enhanced soy Isoflavone bioconversion

Hyperstable β-glucosidase engineered via semi-rational design shows 4.6-fold longer half-life. • T47A mutant achieves dual thermostability enhancement and 44.4% efficiency boost. • Flexible loop rigidification near active site revealed as novel stabilization mechanism. • High glucose tolerance ( K i  = 3.72 mM) maintained for efficient isoflavone conversion. The thermal instability of β-glucosidases frequently impedes their industrial deployment in soy isoflavone bioconversion. Herein, we engineered a hyperstable Bgl1269 variant via a semi-rational strategy integrating evolutionary conservation analysis, homology modeling, and FoldX free energy calculations. Among six constructed mutants, T47A demonstrated exceptional thermostability, exhibiting a 4.6-fold extended half-life at 45 °C (1.64 ± 0.03 h), a 5 °C elevated optimal temperature, and an increased T50 value (51.36 °C). Kinetic analyses revealed enhanced catalytic efficiency for daidzin (44.4% increase) and genistin (39.1% increase), while preserving robust substrate affinity and glucose tolerance ( K i  = 3.72 mM). Molecular dynamics simulations attributed these enhancements to reduced flexibility in active site-adjacent loops, reinforcing structural rigidity without compromising catalytic integrity. This study delivers a potent biocatalyst for industrial isoflavone aglycone production and a generalizable glycosidase thermostabilization paradigm through targeted engineering of non-catalytic loops.