A ginsenosidase specifically hydrolyzing multi-20-O-glycosides of protopanaxadiol type ginsenosides such as ginsenoside Rb(1), Rb(3), Rb(2) and Rc, named ginsenosidase type II, was isolated and purified from Aspergillus sp.g48p strain. The molecular weight of the enzyme was 60 kDa. Ginsenosidase type II was demonstrated to hydrolyze multi-20-O-glycoside of protopanaxadiol type ginsenoside Rb(1), Rb(3), Rb(2) and Rc; i.e. the ginsenosidase type II hydrolyzes 20-O-beta-glucoside of the ginsenoside Rb(1), 20-O-beta-xyloside of ginsenoside Rb(3), 20-O-alpha-arabinoside(p) of ginsenoside Rb(2) and alpha-arabinoside(f) of ginsenoside Rc to produce mainly ginsenoside Rd, and small amount of Rg(3). However, it did not hydrolyze 3-O-beta-glucosides of ginsenoside Rb(1), Rb(3), Rb(2) and Rc which was different with the ginsenosidase type I previously reported, either did not hydrolyze the glycosides of protopanaxatriol type ginsenoside such as ginsenoside Re, Rf and Rg(1), showing significant difference from all previously described glycosidases. (C) 2009 Published by Elsevier Ltd.