Surface Charge Engineering Unlocks Dual Enhancement of Thermostability and Catalytic Efficiency in a Bacterial β-Glucosidase for Sustainable Resveratrol Production.

Resveratrol production via enzymatic hydrolysis of polydatin is hindered by the thermal instability of β-glucosidases. This study employed rational design to engineer Bacillus sp. D1-derived BglD2 through surface charge optimization. The D156E mutant achieved a 42.7% increase in catalytic efficiency (kcat/Km = 49.76 s-1·mM-1) toward polydatin and a 2.1-fold longer half-life at 40 °C. The D411E mutant exhibited a 5.2-fold improvement in thermostability while retaining native activity. Structural analyses revealed that D156E formed a stabilizing salt bridge (2.7 Å) with Lys111 and a hydrogen bond with Pro112, while D411E established compensatory hydrogen bonds with Phe408. Both mutants maintained broad pH activity (pH 6.0-7.0), high glucose tolerance (Ki > 55 mM), and metal ion resilience. This work demonstrates that targeted surface charge engineering concurrently enhances thermostability and catalytic efficiency, enabling efficient resveratrol production with reduced enzyme consumption in industrial applications.