Purification, biochemical and biophysical characterization of lysosomal β-D-glucuronidase from an edible freshwater mussel, Lamellidens corrianus.

A lysosomal glycosidase, β-glucuronidase, has been purified to homogeneity, from the soluble extracts of a freshwater mussel, L. corrianus, by a series of chromatography techniques involving phenyl-Sepharose, ion exchange, affinity and gel filtration chromatography. In native PAGE, β-glucuronidase resolved into a single band and the molecular mass determined by gel filtration chromatography was found to be 250 kDa. Zymogram analysis with 4-methyl umbelliferyl β-glucuronide substrate validated the purified enzyme as β-glucuronidase. In SDS-PAGE, the purified enzyme was resolved into four sub-units with molecular weights around 90, 75, 65, and 50 kDa, respectively, and two of the subunits (90 and 50 kDa) cross-reacted with human β-glucuronidase antiserum. The optimum pH and temperature of the purified glycosidase were 5.0 and 70 °C, respectively. The enzyme kinetics parameters, substrate affinity (K) and maximum velocity (V) of the purified protein estimated with p-nitrophenyl β-D-glucuronide were 0.457 mM and 0.11867 μmol min mL, respectively. The secondary structure of β-glucuronidase was determined in the far-UV range (190 nm to 230 nm) using CD spectroscopy. Heat denaturation plots determined by CD spectroscopy showed that the purified enzyme was stable up to 70 °C.