Glycyrrhizin (GL), the principal sweet-tasting bioactive ingredient of licorice (root of Glycyrrhiza glabra), shows poor oral absorption and gut microbial transformation of GL to glycyrrhetinic acid (GA) plays a major role for its multiple pharmacological effects. Co-administration of GL-hydrolyzing bacteria appears to be a feasible strategy to enhance GA exposure. This study reported a gut bacterial strain Staphylococcus pasteuri 3I10 which exhibited moderate p-nitrophenyl-beta-D-glucuronide (PNPG)-hydrolyzing activity but low GL deglucuronidation activity in its crude lysate. The gus gene encoding S. pasteuri 3I10 beta-glucuronidase was successfully cloned and overexpressed in Escherichia coli BL21(DE3). The purified beta-glucuronidase (SpasGUS) was 71 kDa and showed optimal pH and temperature at 6.0 and 50 degrees C, respectively. Comparing to E. coli beta-glucuronidase (EcoGUS), SpasGUS displayed lower velocity and affinity to PNPG hydrolysis (V-max 16.1 +/- 0.9 vs 140.0 +/- 4.1 mu molmin(-1) mg(-1); K-m 469.4 +/- 73.4 vs 268.0 +/- 25.8 mu M), but could selectively convert GL to GA at much higher efficiency (V-max 0.41 +/- 0.011 vs 0.005 +/- 0.002 mu molmin(-1) mg(-1); K-m 116.9 +/- 15.4 vs 53.4 +/- 34.8 mu M). Molecular docking studies suggested SpasGUS formed hydrogen bond interactions with the glucuronic acids at Asn414, Glu415 and Leu450, and Val159, Tyr475, Ala368, and Phe367 provided a hydrophobic environment for enhanced activity. Two special substrate interaction loops near the binding pocket of SpasGUS (loop 1 beta-glucuronidase) may account for the selective and efficient bioconversion of GL to GA, predicting that loop 1 beta-glucuronidases show high possibility in processing GL than mini-loop 1 and loop 2 beta-glucuronidases. These findings support potential applications of SpasGUS in cleaving GL to facilitate GA production in vivo or in pharmaceutical industry.