Novel Sucrose Phosphorylases Revealing Dynamic Regioselectivity and Their Application in the Synthesis of Glyceryl Glucoside

Glyceryl glucoside (GG) synthesis is constrained by the limited diversity and moderate thermostability of known sucrose phosphorylases (SPases). In this study, we identified six novel SPase-encoding genes by integrating bioinformatics mining, homology modeling, and enzymatic validation. Following heterologous expression in the thermotolerant chassis organism <i>Bacillus licheniformis</i>, functional characterization revealed that SPase from <i>Thermanaerothrix solaris</i><i>4228-RoL</i> (TSP) exhibits an optimal reaction temperature of 70 °C─the highest whole-cell catalytic temperature reported for SPases to date. Furthermore, TSP's product regioselectivity was dynamically tunable by reaction parameters, with the 1-αGG proportion increasing from 6.97% to 58.70% under varying conditions of the glycerol-to-sucrose ratio, substrate concentration, and temperature. Additionally, BsSP from <i>Bifidobacterium scardovii</i><i>JCM 12489</i> achieved a GG yield of 263.86 g/L with 84.28% sucrose conversion under optimized conditions. This study provides a high-performance enzyme toolbox and efficient process for GG synthesis while deepening the mechanistic understanding of SPase catalysis, establishing a foundation for green biomanufacturing.