Biotechnology and Bioprocess Engineering 2021; 26(6): 956-967  
Production of the Minor Ginsenoside F2 from the PPD-mix-type Major Ginsenosides Using a Novel Recombinant Glycoside Hydrolase from Novosphingobium aromaticivorans
Muhammad Zubair Siddiqi, Hye Yoon Park, Ga-Ryun Kim, Chang-Hao Cui, Young Jun Jo, Sun-Chang Kim, and Wan-Taek Im
Muhammad Zubair Siddiqi, Wan-Taek Im*
Department of Biotechnology, Hankyong National University, Anseong 17579, Korea
Tel: +82-31-6705335; Fax: +82-31-6705339
E-mail: wandra@hknu.ac.kr
Muhammad Zubair Siddiqi, Wan-Taek Im
AceEMzyme Co., Ltd., Academic Industry Cooperation, Anseong 17579, Korea
Hye Yoon Park, Ga-Ryun Kim
National Institute of Biological Resources, Incheon 22689, Korea
Chang-Hao Cui, Young Jun Jo, Sun-Chang Kim
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Korea
Received: July 7, 2020; Revised: October 10, 2002; Accepted: January 6, 2021; Published online: December 31, 2021.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: The minor ginsenosides such as F2, C-K, Rh2, and Rg3 make up less than 1% of the total ginseng extract; however, many studies have shown that ginsenoside F2 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 F2. 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 (Rb1, Rb2, Rc, and Rd) into the minor ginsenoside F2. 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 Km value of p-nitrophenyl-β-D-glucopyranoside was 9.06 ± 0.28 and the Vmax value was 24.0 ± 0.34 μmol/min/mg of protein. For gram-scale production of minor ginsenoside F2, crude PPD-mix-type ginsenosides (2 g/400 mL) were treated with BglNar and 1.14 g of F2 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 F2 using a recombinant enzyme will enhance the health benefits of Panax ginseng. This is the first study describing the gram-scale production of F2 from PPD-mix using a single novel ginsenosidetransforming β-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 F2, while in this study we found the possibility of gram-scale production of F2 from PPD-mixtype major ginsenosides using a single recombinant enzyme, thus simplifying the production and reducing the cost.
Keywords: biotransformation, β-glucosidase, recombinant enzyme, ginsenoside F2, Novosphingobium aromaticivorans


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