Partial purification and characterization of three ginsenoside-metabolizing beta-glucosidases from Pythium irregulare

The ginseng pathogen Pythium irregulare is able to selectively metabolize the 20(S) protopanaxadiol ginsenosides Rb1, Rb2, Rc, Rd, and gypenoside XVII via extracellular glycosidases, leading to the formation and partial assimilation of ginsenoside F2. Herein we have partially purified three ginsenoside-deglycosylating enzymes from P. irregulare culture filtrates, and provide preliminary characterization. A protocol involving acetone precipitation, chromatofocusing on PBE 94, gel filtration on Sephacryl S-200 HR and ion-exchange on Q Sepharose Fast Flow resulted in a 13-25-fold purification. The three enzymes were induced in cultures grown in the presence of ginsenosides, and found to be acidic proteins (pI of 4.5-5.0), consisting of an apparent high molecular weight (approximately 160 kDa) homodimer of 78 kDa subunits, with beta(1-->6) activity, and two monomeric enzymes of 61 and 57 kDa, with beta(1-->2) activity. Primary sequence analysis identified them as beta-glucosidases, with no homology to other saponin-deglycosylating enzymes. These are the first glycosidases purified from a Pythium species. We speculate that their role is likely to help Pythium find its host, and/or obtain nutrients/growth factors from its environment.