Structure-Guided Engineering of a Sucrose Phosphorylase from <i>Clostridium beijerinckii</i> for Efficient α-Arbutin Production

α-Arbutin is widely used in pharmaceutical and cosmetic products. We identified and engineered a novel sucrose phosphorylase (SPase) from Clostridium beijerinckii (CbSP). A double variant CbSPM2 (A197P/V60T) was constructed via structure-guided and HotSpot Wizard analysis strategies, which exhibited a 78-fold increase over the wild-type CbSP (CbSP-WT) in catalytic efficiency, along with an extension of half-life by 3.16 h at 40 °C. Mechanistic analysis revealed that the A197P mutation reshaped the hydrogen bond network and hydrophobic interaction around the active center, while the V60T mutation optimized surface polar interactions and formed additional polar contacts with the surrounding solvent molecules. Molecular dynamics (MD) simulations and dynamic cross-correlation matrix (DCCM) analysis demonstrated these changes enhanced substrate binding, reduced flexibility, and strengthened conformational rigidity. The engineered strain E. coli::CbSPM2 produced 122.7 g/L α-arbutin within 24 h under a nitrogen-protected system via batch feeding with 55 g/L hydroquinone (HQ), achieving a substrate conversion rate of 90.23%─the highest whole-cell catalytic yield reported. Collectively, this study contributes an efficient biocatalyst and a scalable green process for α-arbutin production, laying a solid foundation for industrial manufacturing of α-arbutin both with efficiency and the principles for green chemistry.