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Master of Science




Mark Bernards


A putative ginsenosidase (PiGH1-x) from the oomycete pathogen of American Ginseng, Pythium irregulare, was expressed in E. coli DH5α to assess its capability to partially deglycosylate ginsenosides. The recombinant protein was extracted using non-denaturing and denaturing conditions and purified using gel filtration chromatography and nickel affinity purification. Glycosidase activity was tested using p-nitrophenyl β-D-glucopyranoside (pNPG) as the substrate and measured through spectrophotometry. Ginsenosidase activity was tested using ginsenoside Rb1 as the substrate, reaction products were identified using LC-MS. Recombinant PiGH1-x cleaved glucose from pNPG, and converted ginsenoside Rb1 into Rd and Gypenoside XVII, demonstrating both (12) and (16) glycosidase activity. While a high yield of recombinant protein was observed, protein aggregation interfered with the purification process, and further optimization is required. Since previous work has correlated ginsenoside deglycosylation with infection severity, these findings suggest that PiGH1-x may have an important role in the pathogenicity of P. irregulare towards American Ginseng.

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