A novel beta-glucosidase from Penicillium aculeatum was purified as a single 110.5-kDa band on SDS-PAGE with a specific activity of 75.4 U mg(-1) by salt precipitation and Hi-Trap Q HP and Resource Q ion exchange chromatographies. The purified enzyme was identified as a member of the glycoside hydrolase 3 family based on its amino acid sequence. The hydrolysis activity for p-nitrophenyl-beta-d-glucopyranoside was optimal at pH 4.5 and 70 A degrees C with a half-life of 55 h. The enzyme hydrolyzed exo-, 3-O-, and 6-O-beta-glucosides but not 20-O-beta-glucoside and other glycosides of ginsenosides. Because of the novel specificity, this enzyme had the transformation pathways for ginsenosides: Rb-1 -> aEuro parts per thousand Rd -> aEuro parts per thousand F-2 -> aEuro parts per thousand compound K, Rb-2 -> aEuro parts per thousand compound O -> aEuro parts per thousand compound Y, Rc -> aEuro parts per thousand compound Mc(1) -> aEuro parts per thousand compound Mc, Rg(3) -> aEuro parts per thousand Rh-2 -> aEuro parts per thousand aglycone protopanaxadiol (APPD), Rg(1) -> aEuro parts per thousand F-1, and Rf -> aEuro parts per thousand Rh-1 -> aEuro parts per thousand aglycone protopanaxatriol (APPT). Under the optimum conditions, the enzyme converted 0.5 mM Rb-2,Rb- Rc, Rd, Rg(3), Rg(1), and Rf to 0.49 mM compound Y, 0.49 mM compound Mc, 0.47 mM compound K, 0.23 mM APPD, 0.49 mM F-1, and 0.44 mM APPT after 6 h, respectively.