Purification and characterization of two novel β-D-glucuronidases converting glycyrrhizin to 18β-glycyrrhetinic acid-3-O-β-D-Glucuronide from Streptococcus LJ-22

Two novel β-glucuronidases, which metabolize glycyrrhizin (GL) to 18β-glycyrrhetinic acid-3-O-β-D-glucuronide (GAMG), were purified from Streptococcus LJ-22 isolated from human intestinal microflora. β-Glucuronidases I and II were purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, butyl toyopearl, Q-Sepharose, hydroxyapatite Ultrogel, and GL-attached Sepharose column chromatographies, with the final specific activities of 137 and 190 nmole/min/mg, respectively. The molecular sizes of both β-glucuronidases were found to be 140 kDa by gel filtration, and they consisted of two identical subunits (M.W. 67 kDa by SDS-PAGE). β-Glucuronidases I and II showed optimal activity at pH 7.0 and pH 6.5, respectively. Both purified enzymes were potently inhibited by Cu²⁺ and PCMS, and had maximum activity on glycyrrhizin, but did not hydrolyze p-nitrophenyl-β-glucuronides, baicalin, or GAMG. These findings suggest that the biochemical properties and substrate specificities of these enzymes are different from those of the previously purified β-glucuronidases. This is the first reported purification of sugar (not aglycone)-recognizing β-glucuronidases from intestinal bacteria.