Purification and characterization of a highly selective glycyrrhizin-hydrolyzing β-glucuronidase from Penicillium purpurogenum Li-3

Abstract

A novel β-glucuronidase from filamentous fungus Penicillium purpurogenum Li-3 was purified to electrophoretic homogeneity by ultrafiltration, ammonium sulfate precipitation, DEAE-cellulose ion exchange chromatography, and Sephadex G-100 gel filtration with an 80.7-fold increase in specific activity. The purified β-glucuronidase is a dimeric protein with an apparent molecular mass of 69.72kDa (m/z=69,717), determined by MALDI/TOF-MS. The optimal temperature and pH of the purified enzyme are 40°C and 6.0, respectively. The enzyme is stable within pH 5.0–8.0, and the temperature up to 45°C. Mg2+ ions enhanced the activity of the enzyme, Ca2+ and Al3+ showed no effect, while Mn2+, Zn2+, Hg2+ and Cu2+ substantially inhibited the enzymatic activity. The Km and Vmax values of the purified enzyme for glycyrrhizin (GL) were evaluated as 0.33mM and 59.0mmolmg−1min−1, respectively. The purified enzyme displayed a highly selective glycyrrhizin-hydrolyzing property and converted GL directly to glycyrrhetic acid mono-glucuronide (GAMG), without producing byproduct glycyrrhetic acid (GA). The results suggest that the purified enzyme may have potential applications in bio-pharmaceutical and biotechnological industry.