Production of the Minor Ginsenoside F-2 from the PPD-mix-type Major Ginsenosides Using a Novel Recombinant Glycoside Hydrolase from Novosphingobium aromaticivorans

Abstract

Background: The minor ginsenosides such as F-2, C-K, Rh2, and Rg3 make up less than 1% of the total ginseng extract; however, many studies have shown that ginsenoside F-2 has anti-cancer and antioxidant effects and improves dementia and atopic dermatitis. Therefore, the enhanced production of this minor ginsenoside is a promising approach for the pharmaceutical industry. In this study, we found and cloned a novel glycoside hydrolase gene for the gram-scale production of ginsenoside F-2. Methods and Results: Novosphingobium aromaticivorans, which was isolated from deep-terrestrial-subsurface sediments, has shown ginsenoside-converting ability. From this bacterium, a novel glycoside hydrolase, named BglNar, was found that can efficiently biotransform the protopanaxatriol (PPD-mix-type) major ginsenosides (Rb-1, Rb-2 Rc, and Rd) into the minor ginsenoside F-2. This enzyme was cloned, expressed in Escherichia coli BL21, and characterized. The BglNar comprises 439 amino acid and belongs to the glycoside hydrolase family 1. The K-m value of p-nitrophenyl-beta-D-glucopyranoside was 9.06 +/- 0.28 and the V-max value was 24.0 +/- 0.34 mu mol/min/mg of protein. For gram-scale production of minor ginsenoside F-2, crude PPD-mix-type ginsenosides (2 g/400 mL) were treated with BglNar and 1.14 g of F-2 with final purity of 82.5 +/- 1.3%, was obtained after purification using a column packed with HP20 resin. Conclusion: Our preliminary data demonstrate that the gram production of ginsenoside F-2 using a recombinant enzyme will enhance the health benefits of Panax ginseng. This is the first study describing the gram-scale production of F-2 from PPD-mix using a single novel ginsenoside-transforming beta-glucosidase of the GH family 1. Significance and Impact of Study: In most of the previous studies, researchers have been used the combination of enzymes for production of F-2, while in this study we found the possibility of gram-scale production of F-2 from PPD-mix-type major ginsenosides using a single recombinant enzyme, thus simplifying the production and reducing the cost.