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

摘要

Background Ginsenoside Rh2( S ) 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( S ). Method Three glycoside hydrolases (BglBX10, Abf22-3, and BglSk) were cloned in Escherichia coli BL21 (DE3) and the expressed recombinant enzyme was used for the scaled-up production of Rh2( S ) 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( S )?→?Rh2( S ). 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( S ), and then Rg3( S ) was completely transformed to Rh2( S ) by BglSk. As a result, 15.1?g of ginsenoside Rh2( S ) 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( S ) at the industrial level.