Enhanced production of ginsenoside Rh2() from PPD-type major ginsenosides using BglSk cloned from together with two glycosidase in series.

Background: Ginsenoside Rh2() is a promising compound for the prevention of various kinds of cancers, inflammation, and diabetes. However, due to its low concentration (<0.02%), researchers are still trying to find an efficient glycoside hydrolase for the scaled-up production of Rh2().

Method: Three glycoside hydrolases (BglBX10, Abf22-3, and BglSk) were cloned in BL21 (DE3) and the expressed recombinant enzyme was used for the scaled-up production of Rh2() through the conversion of PPD-type (protopanaxadiol) major ginsenosides (Rb1, Rc, and Rd, except Rb2) extracted from Korean red ginseng. Specific and specialized bioconversion pathways were designed that evolved the initial bioconversion of PPD-mix → Rg3() → Rh2(). The reaction was started with 50 mg/mL of PPD-mix, 20 mg/mL of BglBX10, Abf22-3, and BglSk in series, respectively. The process was completed in a 10 L jar fermenter with a 5 L working volume at 37 °C for 48 hrs.

Results: The designed bioconversion pathways show that Abf22-3 and BglBX10 were responsible for the conversion of Rb1, Rc and Rd → Rg3(), and then Rg3() was completely transformed to Rh2() by BglSk. As a result, 15.1 g of ginsenoside Rh2() with 98.0 ± 0.2% purity was obtained after strict purification using the Prep-HPLC system with a 100 φ diameter column. Additionally, BglSk was also investigated for its production activity with seven different kinds of PPD-mix type ginsenosides.

Conclusion: Our pilot data demonstrate that BglSk is a suitable enzyme for the gram unit production of ginsenoside Rh2() at the industrial level.